A  TEXT-BOOK 


ON 


DISBASE-PEODUCING 


MICKOOKG-ANISMS 


ESPECIALLY  INTENDED 


FOE  THE  USE  OF  VETERINABY  STUDENTS 
AND  PRACTITIONERS 


BY 

MAXIMILIAN  HERZOG,  M.D. 

PROFESSOR    OF    PATHOLOGY   AND    BACTERIOLOGY   IN   THE    CHICAGO    VETERINARY    COLLEGE,    PATHOL- 
OGIST TO  THE  GERMAN  AND  THE  ALEXIAN  BROTHERS'  HOSPITALS  OF  CHICAGO,  LATE 
PATHOLOGIST  IN  THE  BUREAU  OF  SCIENCE,  MANILA,  P.  I.,  ETC. 


WITH   214  ILLUSTRATIONS  IN   BLACK  AND   14  COLORED  PLATES 


LEA  &  FEBIGER 
PHILADELPHIA   AND  NEW   YORK 


BlOLOGt 
R 
G 


Entered  according  to  Act  of  Congress,  in  the  year  1910,  by 

LEA  &  FEBIGER, 
in  the  Office  of  the  Librarian  of  Congress.     All  rights  reserved. 


Main  Lib. , 


PREFACE 


THERE  does  not  exist,  so  far  as  the  author  knows,  in  the  English 
language,  any  text-book  on  Pathogenic  Microorganisms,  written 
especially  for  the  use  of  veterinary  students  and  practitioners,  nor 
are  there  many  such  books  in  any  language.  In  fact,  the  only  work 
of  this  kind  with  which  the  author  has  been  familiar  in  the  past  is 
by  Professor  Th.  Kitt,  Bakterienkunde  und  Pathologische  Mikro- 
scopie  fur  Thierarzte  und  Studierende  der  Thier  Medizin.  While 
lecturing  and  conducting  the  practical  courses  in  pathology  and 
bacteriology  during  the  last  few  years  in  the  Chicago  Veterinary 
College,  the  author  has  been  often  approached  by  students  with  the 
request  to  furnish  them  with  a  manifold  summary  of  the  lectures  and 
laboratory  talks  on  the  use  of  the  different  stains,  culture  methods, 
animal  inoculations,  etc.  It  was  first  during  the  course  of  1909-10 
that  the  author  acceded  to  this  demand  and  furnished  to  the  students 
a  mimeographed  summary  of  the  work  in  these  departments.  The 
result  was  satisfactory  both  to  teacher  and  students,  and  the  author, 
therefore,  concluded  to  prepare  a  systematic  text-book  covering  the 
needs  of  veterinary  students  and  practitioners,  with  illustrations 
wherever  desirable.  He  feels  confident  that  such  a  publication  will 
be  of  assistance  both  to  the  teachers  of  bacteriology  in  veterinary 
schools  and  to  their  students,  and  hopes  that  the  book  will  find  an 
interested  circle  of  readers  ana  consultants  among  veterinary  practi- 
tioners. Many  of  these  have  been  students  when  bacteriology  was 
very  insufficiently  taught  in  most  of  the  veterinary  schools  of  our 
country,  and  now,  when  the  importance  of  this  subject  has  been  so 
well  recognized  both  in  human  and  in  veterinary  medicine,  they 
cannot  help  but  feel  the  necessity  of  getting  an  elementary  knowledge 
of  the  theory  and  practice  of  dealing  with  pathogenic  microorganisms. 
In  the  consideration  of  the  infectious  diseases  of  animals  most  impor- 
tant in  veterinary  practice,  the  morbid  anatomy  and  histopathology 
has  also  been  fully  covered  in  the  following  pages.  It  has  been  the 
constant  endeavor  of  the  author  to  be  explicit  and  to  introduce  and 
develop  the  subject  in  such  a  manner  that  the  book  might  be  used  for 
self-instruction  by  any  reader  who  has  already  gained  a  moderate 
elementary  knowledge  of  biology. 

While  the  book  is  primarily  intended  for  veterinary  students  and 
practitioners,  it  is  hoped  that  it  will  also  be  of  use  to  the  medical  student 

222586 


iv  PREFACE 

who  wishes  to  give  attention  to  the  comparative  bacteriology  of  man 
and  the  domestic  animals.  It  may  also  find  a  place  in  the  curriculum 
of  agricultural  colleges  where  bacteriology  is  becoming  more  and 
more  taken  up,  and  where  it  is  also  especially  studied  with  reference 
to  veterinary  science  and  to  certain  fermentative  processes  in  the 
soil,  and  in  milk  and  milk  products,  as  butter,  cheese,  etc.  Micro- 
organisms in  relation  to  such  processes  have,  therefore,  been  fully 
considered. 

A  somewhat  novel  feature  for  a  book  of  this  character  has  been 
introduced,  namely,  the  addition  after  each  subject  or  chapter  of 
"Questions."  A  student  may  read  a  chapter  once  or  even  several 
times,  and  he  may  think  that  he  has  fully  mastered  and  well  remembers 
the  subject.  Yet  when  it  comes  to  exercises  in  recitation  in  the  class 
room  or  to  a  written  examination  he  may  fail.  The  author,  therefore, 
believes  that  it  will  be  of  great  advantage  to  the  student  to  have  after 
each  chapter  a  number  of  questions  covering  the  subject  treated, 
and  enabling  him  to  put  his  knowledge  to  a  test  to  find  out  whether 
or  not  he  has  mastered  the  task  of  committing  to  memory  the  main 
facts  given.  Such  questions  in  a  voluminous  text-book  or  work  of 
reference  would,  of  course,  be  unnecessary,  but  they  will  serve  a  useful 
purpose  in  an  elementary  book  for  the  beginner,  whether  he  be 
student  or  practitioner. 

The  author  has  freely  consulted  the  following  works:  Kolle  and 
Wassermann,  Handbuch  der  Pathogenen  Microorganismen;  Kitt, 
Eakterienkunde  und  Pathologische  Mikroscopie  fur  Thierdrzte;  Fliigge, 
Die  Mikroorganismen;  Nocard  and  Leclainche,  Les  Maladies  Micro- 
biennes  des  Animaux;  Moore,  The  Pathology  and  Differential  Diag- 
nosis of  Infectious  Diseases  of  Animals;  Hutyra  and  Marek,  Specielle 
Pathologic  und  Therapie  der  Hausthiere;  Laveran,  Trypanosomes 
and  Trypanosomiases;  Doflein,  Die  Protozoen  als  Parasiten  und 
Krankheitserreger;  Calkins,  Protozoology;  Lafar,  Technische  Myko- 
logie;  Kraus  and  Levaditi,  Handbuch  der  Technik  und  Methodik  der 
Immunitdtsforschung;  the  publications  of  the  Bureau  of  Animal 
Industry,  United  States  Department  of  Agriculture,  and  those  of  the 
Bureau  of  Science,  Manila,  P.  I. 

M.  H. 

CHICAGO,  1910. 


CONTENTS 


PAET  I 

THE  THEORY  AND  PRACTICE  OF  GENERAL 
BACTERIOLOGY 

CHAPTER  I 
INTRODUCTORY  HISTORICAL  REVIEW 17 

CHAPTER  II 

ORGANISMS — SAPROPHYTES — PARASITES — GENERAL  REMARKS  ON  DISEASE- 
PRODUCING  MICROORGANISMS 22 

CHAPTER  III 
BACTERIA — GENERAL  CONSIDERATIONS — MORPHOLOGY 26 

CHAPTER  IV 
BIOLOGY  OF  BACTERIA 38 

CHAPTER  V 
OCCURRENCE  OF  BACTERIA  IN  NATURE — ROUTES  OF  ENTRANCE  IN  INFECTION    47 

CHAPTER  VI 
INFECTION — PHAGOCYTOSIS — OPSONINS ;  53 

CHAPTER  VII 

ANTIBODIES — IMMUNITY — EHRLICH'S  SIDE-CHAIN  THEORY — THE  WASSER- 

MANN  SERUM  TEST gg 

CHAPTER  VIII 

METHODS  OF  OBSERVING  BACTERIA— THE  USE  OF  THE  MICROSCOPE  AND 

ACCESSORIES §7 


vi  CONTENTS 

CHAPTER  IX 
STAINING  OF  BACTERIA  IN  COVER-GLASS  PREPARATIONS  AND  IN  TISSUES          1( 

CHAPTER  X 
CULTURE  MEDIA  AND  THEIR  STERILIZATION    . 


CHAPTER  XI 

CULTURE  MEDIA  IN  RELATION  TO  METABOLIC  PRODUCTS — TESTS  FOR  THE 
LATTER  . 


CHAPTER  XII 

METHODS  OF  OBTAINING  PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL  AND 
OTHER  SOURCES     ..........     

CHAPTER  XIII 

IDENTIFICATION     OF    BACTERIA — CULTURAL    CHARACTERISTICS — ANIMAL 

EXPERIMENTS 1» 

CHAPTER  XIV 

METHODS  OF  EXAMINING  BACTERIA — IN  AIR — SOIL— WATER  AND  OTHER 
FLUIDS 

CHAPTER  XV 
PRINCIPLES  OF  DISINFECTION — DISINFECTANTS .     .     If 


PART  II 

SPECIAL  BACTERIOLOGY  AND  THE  HIGHER  VEGE-£ 
TABLE  PATHOGENIC  ORGANISMS. 

CHAPTER  XVI 
WOUND  INFECTION— SUPPURATION  AND  THE  COMMON  PYOGENIC  BACTERIA     193 

CHAPTER  XVII 

PYOGENIC  BACTERIA  IN  DOMESTIC  ANIMALS— STREPTOCOCCUS  EQUI— STREP- 
TOCOCCUS IN  MORBUS  MACULOSUS  EQUI  AND  PLEURO  PNEUMONIA  ID 
HORSES— BOTRYOCOCCUS  ASCOFORMANS— PYOGENIC    BACTERIA   IN      ^ 
CATTLE — BACILLUS  PYOGENES  Suis 20< 


CONTENTS 


vn 


CHAPTER  XVIII 

BACTERIA  PRODUCING  DIPHTHERITIC  INFLAMMATIONS— BACILLUS    DIPH- 
THERIA—BACILLUS NECROPHORUS— BACILLUS  DIPHTHERIA  AVIUM 


CHAPTER  XIX 
BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP— BACILLUS  AVISEPTICUS 

BOVISEPTICUS — OVISEPTICUS SuiSEPTICUS   AND   EQUISEPTICUS 

BACILLUS    OF    DOG    TYPHOID— PLAGUE    BACILLUS    IN   MAN   AND 
ANIMALS 

CHAPTER  XX 

941 

ANTHRAX  BACILLUS 

CHAPTER  XXI 
BACILLUS  OF  SYMPTOMATIC  ANTHRAX 254 

CHAPTER  XXII 

BACILLUS  OF  MALIGNANT  EDEMA  AND  SIMILAR  BACTERIA — BACILLUS  OF 
GASTROMYCOSIS  Ovis — BACILLUS  AEROGENES  CAPSULATUS  .      .      .     263 

CHAPTER  XXIII 
BACILLUS  OF  TETANUS 269 

v 

CHAPTER  XXIV 

BACILLI  OF  THE  TYPHOID-COLON-HOG  CHOLERA  GROUP — BACILLUS  CHOLERA 
Suis — BACILLUS  TYPHOSUS — BACILLUS  COLI  COMMUNIS — WHITE 
SCOURS  IN  CALVES — MALIGNANT  CATARRH  OF  CATTLE — BACTERIUM 
PHLEGMASIA  UBERIS — BACILLUS  TYPHI  MURIUM — DANYSZ'S  BA- 
CILLUS— PSITTACOSIS— BACTERIUM  PULLORUM 277 

CHAPTER  XXV 

BACILLUS  OF  SWINE  ERYSIPELAS — BACILLUS  OF  MOUSE  SEPTICEMIA      .      .     298 

CHAPTER  XXVI 
GLANDERS  BACILLUS   .  304 


CHAPTER  XXVII 

BACILLUS  OF  INFECTIOUS  ABORTION — STREPTOCOCCUS  IN   ABORTION    IN 

MARES — STREPTOCOCCUS  OF  VAGINITIS  VERRUCOSA  OF  CATTLE  .  318 


viii  CONTENTS 

CHAPTER  XXVIII 

TUBERCULOSIS — DISTRIBUTION  AMONG  MAN  AND    ANIMALS— ROUTES  -OF 

INFECTION 224 

_ 

CHAPTER  XXIX 

TUBERCULOSIS  (CONTINUED) — HISTOPATHOLOGY  AND  MORBID  ANATOMY  IN 

MAN  AND  ANIMALS 331 

CHAPTER  XXX 

TUBERCULOSIS  (CONTINUED) — THE  BACILLUS  OF  TUBERCULOSIS — THE 
TUBERCULIN  TESTS — BOVOVACCINE — THE  INTERTRANSMISSIBILITY 
OF  BOVINE  AND  HUMAN  TUBERCULOSIS — AVIAN  TUBERCULOSIS  .  346 

.      CHAPTER  XXXI 

PSEUDOTUBERCULOSIS  AND  AdD-FAST  BACILLI  OTHER  THAN  THE  TUBERCLE 
BACILLUS — RAT  LEPROSY — CHRONIC  BACTERIAL  DYSENTERY — 
JOHNE'S  DISEASE  IN  CATTLE 366 

CHAPTER  XXXII 

VARIOUS  Cocci  PATHOGENIC  FOR  DOMESTIC  ANIMALS  AND  MAN — DIPLO- 
coccus  MENINGITIDIS  EQUI — DIPLOCOCCUS  INTRACELLULARIS — DIP- 
LOCOCCUS  PNEUMONIA — MICROCOCCUS  CATARRH  ALIS — PNEU  MO- 
COCCUS  OF  FRIEDLANDER — GONOCOCCUS — MICROCOCCUS  CAPRINUS — 
MICROCOCCUS  MELITENSIS  ..:...  .^ 370 

CHAPTER  XXXIII 

SPIRILLA  —  PATHOGENIC  VIBRIONES  —  SPIROCHETE  —  THE  VIBRIONES  OF 
CHICKEN  SEPTICEMIA  AND  ASIATIC  CHOLERA— SPIROCHETE  IN  MAN, 
OTHER  MAMMALS,  AND  BIRDS .  383 

CHAPTER  XXXIV 
THE  BACILLUS  LACTIMORBI  OF  TREMBLES       .....  .     .     393 

CHAPTER  XXXV 

A  LOCAL  EQUINE  DISEASE  AND  A  BACILLUS  OF  THE  SUBTILIS  GROUP     .     .     396 

CHAPTER  XXXVI 

LOWER     HYPHOMYCETES — TRICHOMYCETES — LEPTOTHRIX — CLADOTHRIX — 

STREPTOTHRIX  AND  ACTINOMYCES 398 

CHAPTER  XXXVII 

ACTINOMYCOSIS 


CONTENTS  ix 

CHAPTER  XXXVIII 

HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE — LEECHES  OR  BUR- 
SATTEE  — PNEUMONOMYCOSIS  —  DERMATOMYCOSIS  —  TRICHOPHYTON 

TONSURANS — ACHORION    SCHONLEINII — FuSARIUM     EQUINORUM 

OIDIUM  ALBICANS  .  416 


CHAPTER  XXXIX 

BLASTOMYCES  —  EPIZOOTIC  LYMPHANGITIS  IN  HORSES  —  BLASTOMYCOTIC 

DERMATITIS  426 


CHAPTER  XL 

BACTERIA,  GENERALLY  NOT  PATHOGENIC,  OFTEN  EMPLOYED  IN  LABORATORY 
PRACTICE — BACILLI  OF  THE  PROTEUS  GROUP — BACILLUS  ANTHRA- 
COIDES  —  BACILLUS  MEGATHERIUM  —  BACILLUS  PRODIGIOSUS  — 
BACILLUS  VIOLACEUS  —  BACILLUS  CYANOGENUS  —  MICROCOCCUS 
TETRAGENUS — MICROCOCCUS  AGILIS — SARCINA  LUTEA  ....  434 


CHAPTER  XLI 

INFECTIOUS    DISEASES    DUE  TO  ULTRAMICROSCOPIC  VIRUSES  —  PLEURO- 

PNEUMONIA   IN    CATTLE  CATTLE    PLAGUE  HOOF-AND-MOUTH 

DISEASE  439 


PAET  III 

MICROORGANISMS  IN  FOODS  AND  SOILS. 

CHAPTER  XLII 

THE  BACTERIA  OF   MEAT-POTSONTNG — BACILLUS  ENTERITIDIS — BACILLUS 

BOTUUNUS 451 

CHAPTER  XLIII 

BACTERIA  OF  THE  NITROGEN  CYCLE — FERMENTATION  OF  UREA — NITRIFYING 

AND  DENTRIFYING  ORGANISMS — FREE  NITROGEN  FIXATION      .     .     457 

CHAPTER  XLIV 

ACETIC-ACID  BACTERIA  466 


x  CONTENTS 

CHAPTER  XLV 

THE  BACTERIOLOGY  AND  BACTERIOLOGIC  EXAMINATION  OF  MILK — GENERAL 
INTRODUCTORY  CONSIDERATIONS — THE  CHANGE  OP  LACTOSE  INTO 
LACTIC  ACID — LACTIC-ACID  BACTERIA — ANAEROBIC  BUTYRIC-ACID 
FORMERS — PEPTONIZING  BACTERIA — ALCOHOLIC  FERMENTATION  OP 
MILK  .  469 


CHAPTER  XLVI 

THE  BACTERIOLOGY  AND  THE  BACTERIOLOGIC  EXAMINATION  OP  MILK 
(CONTINUED)— PATHOGENIC  BACTERIA  IN  MILK— THE  TUBERCLE 
BACILLUS — METHODS  FOR  DETERMINING  ITS  PRESENCE  IN  MILK — 
HUMAN  AND  OTHER  CATTLE  DISEASES  TRANSMISSIBLE  THROUGH 
MILK — NUMBER  AND  SIGNIFICANCE  OP  LEUKOCYTES  IN  MILK  481 


CHAPTER  XLVII 

THE  BACTERIOLOGY  AND  THE  BACTERIOLOGIC  EXAMINATION  OP  MILK 
(CONTINUED) — QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK — 
INTERPRETATION  OP  THE  RESULTS  OP  BACTERIAL  COUNTS  IN 
MILK  —  DETERMINATION  OP  THE  ACIDITY  OF  MILK  —  CERTIFIED 
MILK — PASTEURIZATION  OP  MILK — ITS  ADVANTAGES  AND  DISAD- 
VANTAGES— STORCH'S  TEST  .  494 


CHAPTER  XLVIII 
BACTERIA  IN  BUTTER  AND  CHEESE-MAKING 513 

CHAPTER  XLIX 

SIMPLE  CHEMICAL  MANIPULATIONS — NORMAL  SOLUTIONS  AND  INDICATORS 

REQUIRED  IN  LABORATORY  WORK  IN  BACTERIOLOGY     ....     520 


PART  IV 
PATHOGENIC  PROTOZOA. 

CHAPTER  L 

GENERAL  CONSIDERATION  OP  PROTOZOA — CLASSIFICATION — MORPHOLOGY 

AND  REPRODUCTION    .     .     ....     .     .     .     .     .     .     .     .     529 

CHAPTER  LI 

AMEBA — CLASSIFICATION  AND  MORPHOLOGY — CULTIVATION — PATHOGENIC 

AMEBA — ENTEROHEPATITIS  IN  TURKEYS — BALANTIDIUM  COLI          .     539 


CONTENTS  xi 

CHAPTER  LII 

TRYPANOSOMES    AND    TRYPANOSOMIASES — CERCOMONAS— TRICHOMONAS — 

HERPETOMONAS 553 

CHAPTER  LIII 

PATHOGENIC    SPOROZOA  —  COCCIDIA  —  HEMOSPORIDIA  —  MICROSPORTDIA  — 

SARCOSPORIDIA 568 

CHAPTER  LIV 

PlROPLASMA  BOVIS — TEXAS  FEVER  AND  PlROPLASMOSES  IN  OTHER  ANIMALS       581 

CHAPTER  LV 

RABIES  AND  THE  NEGRI  BODIES  (NEURORYCTES  HYDROPHOBIA)       .  597 


APPENDIX 

THE  METRIC  SYSTEM — THERMOMETRIC  SCALES     .      .      .  621 


PART    I. 

THE  THEORY  AND  PRACTICE  OF  GENERAL 
BACTERIOLOGY. 


CHAPTER    I. 

INTRODUCTORY  HISTORICAL  REVIEW. 

THE  sciences  developed  by  mankind  owe  their  early  awakening 
and  subsequent  growth  to  two  entirely  different  sets  of  motives.  One 
of  these  is  furnished  by  the  necessities  of  life,  in  its  everlasting  struggle 
for  existence;  the  other  by  that  intense  desire  of  the  human  race  to 
unravel  the  mysteries  of  nature  and  solve  the  enigma  of  the  origin 
of  life. 

That  side  of  modern  medical  science  which  deals  with  micro- 
organisms in  relation  to  disease  begins  with  attempts  to  recognize  the 
true  nature  and  cause  of  disease  and  with  experiments  to  ascertain 
whether  life  can  originate  by  spontaneous  generation .  The  conception 
that  many  'diseases  are  due  to  microorganisms  originating  in  or  in- 
vading the  body,  and  multiplying  therein,  was  first  formulated  long 
ago,  to  be  forgotten,  and  to  be  taken  up  again  with  renewed  vigor 
after  the  discovery  that  certain  fermentative  processes  are  due  to  these 
minute  bodies.  This  discovery  wa's  itself  stimulated  by  the  long- 
continued  experimental  quarrel  over  the  question  whether  or  not 
spontaneous  generation  of  life  occurred  in  fermenting  and  putrefying 
organic  materials. 

That  diseases  of  mankind  might  be  due  to  forms  of  life  so  small 
as  to  be  invisible  was  conceived  as  a  purely  hypothetical  idea  long 
before  the  compound  microscope  had  been  invented.  Varo,  in  the 
first  century  before  Christ,  stated  in  writing  that  there  might  perhaps 
exist  animals  so  small  that  they  could  not  be  seen,  but  that  might 
enter  the  human  body  with  the  air  through  the  mouth  and  hose  and 
so  produce  disease.  Nothing,  of  course,  but  a  mere  hypothesis  in 
this  direction  could  be  formed  before  these  microorganisms  were  seen. 

The  first  combination  of  lenses  was  constructed  by  Hans  and 
Zacharias  Janssen,  father  and  son,  living  in  Holland  in  1590.  Their 
2 


18 


INTRODUCTORY  HISTORICAL  REVIEW 


instrument  was  evidently  very  poor  and  did  not  lead  to  any  important 
discoveries.  It  was  not  until  the  seventeenth  century  that  the  micro- 
scope really  gained  importance,  when  Antony  Van  Leewenhoeck,  the 


FIG.  1 


Experiment  of  Schulze:     Forcing  air  through  sulphuric  acid.    (Lafar.) 

true  father  of  microscopy,  succeeded  in  producing  a  fairly  good  instru- 
ment with  magnification  up  to  150  diameters.  He  discovered  the 
spermatozoa,  and  numerous  small  live  organisms  in  saliva,  stagnant 
water,  fermenting  and  decomposing  fluids,  and  organic  materials. 

FIG.  2 


Experiment  of  Schwann:     Heating  air  to  make  it  sterile.     (Lafar.) 

After  these  small  organisms  had  been  studied  for  a  number  of 
years  the  theory  was  promulgated,  particularly  by  Van  Helmont  and 
Needham,  that  these  forms  of  life  arose  by  spontaneous  generation 
in  such  fluids  as  meat  infusion,  etc.,  even  after  they  had  been  boiled. 
This  view  was  contested  by  Spallanzani,  who  showed  that  when  a  meat 
infusion  had  been  boiled  three-quarters  of  an  hour,  and  kept  from 


INTRODUCTORY  HISTORICAL  REVIEW  19 

access  by  the  air  the  development  of  microorganisms  would  not  take 
place.  It  was  then  claimed  by  the  adherents  of  the  theory  of  spon- 
taneous generation  that  the  expulsion  of  the  air  by  boiling  and  the 
arrangements  which  prevented  it  from  reentering  also  prevented 
spontaneous  generation.  Franz  Schulze  and  Theodor  Schwan  then 
devised  methods  to  permit  air  to  enter  after  it  had  either  passed 
through  sulphuric  acid  or  had  been  heated  in  a  glass  tube.  Schwan 
also  showed  that  when  certain  poisonous  chemicals  were  added  to 
the  meat  infusion,  microorganisms  were  not  developed.  His  experi- 
ments were  the  first  to  show  the  effects  of  what  we  now  call  anti- 
septics^ upon  microorganisms.  Schroeder  and  Dusch  allowed  air  to 
enter  the  vessels  containing  boiled  organic  substances  through  glass 
tubes  which  had  been  plugged  with  cotton.  This  method  is  now 
universally  used  in  bacteriologic  work  to  protect  sterile  culture  media 

FIG.  3 


Pasteur  bulb. 

or  pure  cultures  from  contamination.  The  experiments  of  the  last 
two  investigators  were,  however,  not  all  successful,  and  development 
sometimes  occurred  in  their  cotton-plugged  glass  vessels.  The 
question  of  spontaneous  generation  was  not  definitely  settled  when 
Pasteur  took  it  up  before  1860.  He  showed,  in  the  first  place,  that 
a  short  boiling  of  an  infusion  of  organic  material  was  not  sufficient 
to  kill  all  microorganisms,  and  that  some  could  evidently  withstand 
the  temperature  of  boiling  water  for  several  hours.  It  was  subse- 
quently shown  by  the  botanist  F.  Cohn  and  by  Robert  Koch  that 
these  resistant  forms  of  microorganisms  were  the  spores  of  bacteria. 

1  The  term  antiseptic  in  its  strict  sense  means  something  which  will  prevent  putrefaction 
or  sepsis  by  inhibiting  the  growth  of  microorganisms,  while  the  term  germicide  designates 
something  that  will  kill  germs  or  microbes.  The  word  disinfectant  is  used  synonymously  with 
germicide.  In  practice  most  substances  which  act  as  antiseptics  in  a  certain  concentration 
will  generally  in  a  stronger  concentration  act  as  disinfectants  or  germicides.  The  term  anti- 
septics, disinfectants,  and  germicides  are  used  quite  indiscriminately,  and  there  is,  indeed, 
between  them  no  real  generic  difference  but  only  one  of  degree. 


20  INTRODUCTORY  HISTORICAL  REVIEW 

Pasteur  further  constructed  a  peculiarly  shaped  glass  receptacle, 
known  now  as  the  Pasteur  bulb,  which  has  a  bent  and  curved  neck 
through  which  air  can  freely  circulate  without,  however,  introducing 
microorganisms.  In  such  bulbs  meat  infusions  which  had  been 
boiled  for  several  hours  did  not  develop  such  growths.  However, 
as  soon  as  the  neck  was  broken  off,  so  that  microorganisms  could 
fall  into  the  bulb,  the  fluid  would  decompose  with  the  appearance  of 
numerous  microbes.  The  question  of  spontaneous  generation  was 
now  settled,  and  it  had  been  shown  that  microorganisms  could  not 
be  so  formed.  In  the  meantime  the  cause  of  alcoholic  fermentation 
of  sugar-containing  fluids  had  been  discovered.  Erxleben,  as  early 
as  1818,  had  made  the  statement  that  this  fermentation  was  due  to 
the  multiplication  and  the  metabolism  of  yeast  cells.  This,  how- 
over,  was  not  proved  until  about  twenty  years  later  by  the  extensive 
ebservations  and  experiments  of  Cagniard-Latour,  Thedor  Schwann 
and  F.  Kuetzing,  who  worked  on  the  problem  simultaneously.  The 
vegetable  nature  of  yeast  cells  had  also  been  recognized,  and  the 
idea  that  diseases  were  due  to  vegetable  and  other  microorganisms 
received  a  new  stimulus.  Athanasius  Kirchner  had  already  expressed 
this  belief  in  1659,  and  it  was  strongly  upheld  in  the  middle  of  the 
eighteenth  century  by  Plenciz  and  Reimarus;  the  former  believing 
that  each  infectious  disease  was  due  to  a  specific  microorganism. 

These  theories  had  been  long  forgotten  at  the  beginning  of  the 
nineteenth  century;  but  after  the  saccharomyces,  or  yeast  cells,  had 
been  recognized  as  the  cause  of  fermentation,  the  microbic  theory  of 
disease  was  again  revived,  particularly  by  the  celebrated  German 
pathologist  and  anatomist  Henle,  who,  in  1840,  declared  himself  in 
favor  of  this  theory.  He  was  cautious  enough,  however,  to  state 
that  it  was  not  sufficient  to  find  microorganisms  in  certain  diseases, 
but  that  they  must  always  be  present  in  such  cases,  and  further,  that 
they  must  be  shown  actually  capable  of  producing  the  disease.  The 
succeeding  years  brought  observations  and  discoveries  which  demon- 
strated the  fact  that  bacteria  are  the  cause  of  certain  diseases.  The 
anthrax  bacillus  was  seen  and  later  inoculated  into  animals  by 
Pollander  and  Davine  (1850-60).  Rindfleisch,  Recklinghausen, 
Waldeyer,  and  Klebs  saw  the  pyogenic  cocci  in  pyemia,  puerperal 
sepsis,  and  wound  infections.  Robert  Koch,  in  1876,  published  his 
researches  on  the  anthrax  bacillus  and  two  years  later  those  on  mouse 
septicemia.  Bollinger,  in  1878,  recognized  the  significance  of  the 
ray  fungus.  About  this  time  Robert  Koch  introduced  the  use  of 
solid  culture  media  foj*  the  purpose  of  isolating  bacteria  and  obtain- 
ing them  in  pure  cultures.  In  1882  he  published  his  researches  on 
the  tubercle  bacillus.  Kitasato  later  showed  how  to  cultivate  the 
anaerobic  tetanus  bacillus,  and  somewhat  earlier  the  first  disease- 
producing  protozoa  had  been  discovered.  Griffith  Evans,  in  1880,  saw 
in  India,  in  the  blood  of  horses,  mules,  and  camels  suffering  from 
surra,  a  motile  microorganism  which  he  described  as  a  spirillum.  He 


INTRODUCTORY  HISTORICAL  REVIEW  21 

succeeded  in  producing  the  disease  in  healthy  horses  and  dogs  by 
inoculating  the  blood  in  which  he  had  seen  these  organisms.  Steele 
confirmed  Evans'  observations,  and  named  this  pathogenic  organism 
Spirocheta  Evansii.  It  is  now  known  as  Trypanosoma  Evansii, 
and  was  the  first  pathogenic  trypanosome  to  be  discovered.  In 
1882  Laveran  discovered  the  plasmodium  malariae,  and  early  in  the 
next  decade  Theobald  Smith  discovered  the  protozoon  piroplasma 
bigeminum  as  the  cause  of  Texas  fever. 

Since  the  fundamental  work  of  Pasteur  and  Robert  Koch,  studies 
of  pathogenic  microorganisms  in  general  and  of  pathogenic  bacteria 
in  particular  have  been  placed  on  a  firm  basis,  and  have  assumed 
the  greatest  importance  in  the  theory  and  practice  of  human  and 
veterinary  medicine. 


CHAPTER    II. 

ORGANISMS— SAPROPHYTES— PARASITES— GENERAL  REMARKS 
ON  DISEASE-PRODUCING  MICROORGANISMS. 

THERE  are  two  classes  of  objects  in  nature,  one  alive  and  animated, 
the  other  one  inanimate.  To  the  latter  belong  rocks,  minerals, 
chemicals,  etc.  If  we  examine  an  object  belonging  to  this  class,  for 
instance  a  piece  of  iron,  we  find  that  any  part  is  similar  in  structure 
to  the  whole  block,  and  has  all  its  properties.  If,  on  the  other 
hand,  we  examine  a  live  object,  in  other  words  a  living  being,  we 
soon  notice  that  there  are  several,  in  fact  many,  parts  unlike  each 
other  in  properties,  and,  of  course,  individually  unlike  the  whole,  and 
that  these  different  parts  or  portions  perform  different  functions. 
Such  different  parts  of  a  living  being  are  called  its  organs,  and  the 
general  scientific  term  for  live  objects  is  organisms.  Modern  studies 
have  shown  that  all  organisms  are  built  up  of  small  component  parts 
called  cells.  Most  organisms  are  composed  of  a  multitude  of  cells, 
but  there  are  many  which  consist  of  only  a  single  cell.  These  are 
called  unicellular  organisms.  Such  very  minute  beings  can  be  seen 
only  with  the  aid  of  the  microscope,  hence  they  are  known  as  micro- 
organisms, and,  more  popularly,  often  as  microbes. 

We  divide  organisms  into  plants  and  animals,  and  an  enumeration 
of  the  differences  between  a  higher  plant,  for  instance  a  tree  and  a 
higher  animal,  and  for  instance  a  horse,  is  easy.  However,  when  we 
come  to  the  unicellular  organisms  of  the  lowest  type  it  is  sometimes 
difficult  to  decide  whether  we  are  dealing  with  a  minute  plant  or  a 
minute  animal.  There  are,  in  fact,  microorganisms  which  are  classi- 
fied as  vegetables  by  some  investigators  and  as  animals  by  others. 

Pathogenic  Microorganisms. — The  following  pages  will  deal  par- 
ticularly with  the  microorganisms,  both  animal  and  vegetable,  which 
may  invade  the  body  of  man  and  domestic  animals,  and  may  there 
multiply,  in  this  manner  becoming  the  cause  of  the  so-called  infec- 
tious diseases.  Such  minute  disease-producing  organisms,  or  patho- 
genic microorganisms,  belong  to  the  various  phyla,  tribes,  classes, 
orders,  and  families.  The  common  feature  which  makes  them  inter- 
esting and  important  to  the  student  and  practitioner  of  human  and 
veterinary  medicine  is  the  fact  that  they  are  the  cause  of  much  disease, 
suffering,  loss,  and  death.  It  is  well  to  point  out  in  the  beginning 
that  while  certain  of  these  pathogenic  microorganisms  cause  disease 
in  man,  and  not  in  the  lower  animals,  as,  for  instance,  the  microbes 
of  typhoid  fever,  leprosy,  syphilis,  etc.,  and  while  others  cause  disease 


MICROORGANISMS  IN  NATURE  23 

in  the  lower  animals  only,  as,  for  instance,  the  microorganisms  of 
Texas  fever  of  cattle,  black-leg  of  cattle,  diphtheria  of  calves,  leg 
and  lip  diseases  of  sheep,  etc.,  many  other  microorganisms  cause 
identical  or  very  similar  diseases  both  in  man  and  the  lower  animals. 
To  these  latter  belong  the  pathogenic  microorganisms  which  produce 
such  common  diseases  as  tuberculosis,  actinomycosis,  glanders, 
tetanus  or  lock-jaw,  inflammations,  suppurations,  blood-poisoning,  etc. 
Hence,  studies  concerned  with  disease-producing  microorganisms  in 
their  relation  to  human  and  to  veterinary  medicine  overlap,  and  the 
general  underlying  principles  and  the  methods  employed  for  the 
elucidation  of  the  subject  are  identical.  It  is,  of  course,  obvious 
that  in  a  book  primarily  designed  for  veterinary  students  and  practi- 
tioners, pathogenic  microorganisms  will  be  taken  up  especially  with 
reference  to  diseases  of  the  domestic  animals.  Reference  to  human 
diseases  wnll  only  be  made  in  a  brief  manner,  in  so  far  as  is  necessary 
to  point  out  sufficiently  certain  common  features  and  to  emphasize 
the  possibilities  and  the  dangers  of  the  transmission  of  diseases  of 
domestic  animals  to  human  beings,  and  vice  versa.  This  is  a  subject 
in  which  the  veterinarian  is  interested  both  from  a  personal  standpoint 
and  on  behalf  of  the  community.  In  other  words  the  modern  scien- 
tific veterinarian  must  be  a  hygienist,  not  merely  for  the  benefit  of 
his  patients,  the  domestic  animals  and  their  owners,  but  also  for 
the  benefit  of  mankind  at  large. 

Non-pathogenic  Microorganisms. — A  limited  number  of  microorgan- 
isms which  are  not  disease-producers,  and  which,  from  a  medical 
standpoint,  are  entirely  harmless,  will  also  be  considered  briefly.  Such 
microorganisms  are  used  in  the  laboratory  training  of  the  student  to 
familiarize  him  with  morphologic  features  and  technical  methods 
early  in  his  studies,  at  a  time  when  it  would  not  be  advisable  to  give 
into  his  unpractised  hands  dangerous,  live,  disease-producing  micro- 
organisms, and  also  because  some  harmless  widespread  bacteria  are 
very  similar  to  certain  pathogenic  bacteria.  The  student  must  learn 
to  distinguish  such  harmless  bacteria,  as,  for  instance,  the  common 
hay  bacillus  or  B.  subtilis,  from  dangerous  pathogenic  bacteria  like 
the  anthrax  bacillus,  because  they  are  very  similar  in  their  morpho- 
logic and  cultural  features. 

Microorganisms  Causing  Fermentation. — Some  microorganisms  which 
are  important  in  producing  fermentative  changes,  both  desirable  and 
undesirable,  in  milk,  cheese,  and  other  organic  materials,  will  also  be 
briefly  considered.  These  are  also  matters  in  which  the  veterinarian 
is  likely  to  be  consulted  and  in  which  he  should  be  at  least  sufficiently 
well  versed  to  form  an  intelligent  valid  opinion. 

Microorganisms  in  Nature. — Microorganisms  are,  of  course,  not  all 
disease  producers;  in  fact,  the  great  majority  of  them  live  in  the 
outside  world  not  merely  a  harmless  but  a  very  useful  existence. 
Certain  classes  are  the  cause  of  necessary  and  useful  fermentative 
processes;  while  others  bring  about  the  decomposition  of  dead  organic 


24  ORGANISMS,  SAPROPHYTES,  PARASITES 

matter  and  split  it  up  into  simple  chemical  compounds,  so  that 
these  can  be  utilized  again  in  building  up  the  higher  plants,  which 
in  their  turn  are  needed  directly  or  indirectly  to  support  the  life  of 
the  higher  animals.  Without  higher  plants,  herbivorous  animals 
could  not  exist,  and  without  the  latter,  the  carnivora  would  perish. 
Certain  microorganisms  are,  therefore,  essential  in  maintaining  the 
cycle  of  life  on  our  planet.  Microorganisms  do  not  always  live  in 
the  general  outside  world.  They  may  be  in  or  on  other  higher  living 
beings,  as  parasites,  and  yet  they  may  not  do  any  harm  to  their  host 
but  may  even  benefit  it  and  be  a  necessary  element  in  its  metabolism 
and  existence.  So  it  is  incorrect  to  consider  all  microbes  as  enemies 
of  mankind  and  domestic  animals.  It  is  true  that  some  micro- 
organisms are  our  greatest  enemies,  but  many  others  are  our  greatest 
friends. 

Saprophytes  and  Parasites. — Most  microorganisms  of  both  vege- 
table and  animal  types  exist  in  the  outside  world.  They  derive  their 
nutrition  from  dead  organic  material,  which  they  split  up  in  their 
metabolism,  and  they  are  known  as  saprophytes.  The  lowest  micro- 
organisms of  a  vegetable  type,  the  saprophytic  bacteria,  exist  almost 
everywhere  on  and  near  the  surface  of  our  earth.  We  find  them  in  the 
air,  in  the  water,  in  the  soil,  on  the  surface  of  the  bodies  of  animals 
and  plants,  in  decaying  substances,  etc.;  they  are,  as  it  is  also  ex- 
pressed, ubiquitous. 

Organisms  which  live  on  or  in  a  living  host,  and  which  utilize 
some  of  its  material  for  their  own  nutrition,  are  known  as  parasites. 
Some  microorganisms  can  live  only  as  parasites,  and  can  never  thrive 
and  multiply  in  the  outside  world,  as,  for  instance,  the  microorganism 
causing  tuberculosis,  the  tubercle  bacillus,  in  its  various  forms. 
Such  organisms  are  called  strict  or  obligate  parasites. 

There  are  other  microorganisms  which  can  exist  both  as  sapro- 
phytes and  as  parasites,  such  as  those  which  cause  anthrax  in  man  and 
animals.  This  anthrax  bacillus  can  exist  and  multiply  on  meadows, 
in  manure,  in  the  ground,  and  can  also  invade  the  blood  of 
animals,  where  it  greatly  increases  in  numbers  and  causes  the  disease 
known  as  anthrax,  or  splenic  fever.  Microorganisms  which  can 
exist  both  as  saprophytes  and  as  parasites  are  called  facultative  para- 
sites or  facultative  saprophytes,  while  those  which  can  live  only  in 
the  oustide  world  and  never  as  parasites  are  called  strict  or  obligate 
saprophytes. 

When  parasites  live  on  the  outside  of  their  host,  they  are  spoken  of 
as  ectogenous  parasites;  when  they  live  inside  the  body,  as  entogenous 
parasites. 

Commensales. — It  must  not  be  supposed  that  all  parasites  are 
harmful;  many  are  perfectly  harmless.  These  are  called  commen- 
sales. 

The  bacterium  known  as  the  colon  bacillus  is  of  this  type  and 
lives  in  the  colon  and  other  parts  of  the  large  intestines  of  man  and 


PLATE   I 

TYPES  OF  MICROORGANISMS  PRODUCING  DISEASES 
IN  ANIMALS. 


I. — Bacillus  anthracis,  sporulating;  pure  culture  on  agar,  stained  with 
fuchsin.  The  cause  of  anthrax  in  cattle  and  other  domestic  animals. 

II. — Bacillus  sarcophysematos  bo  vis,  sporulating;  pure  culture  on 
glucose  agar,  stained  with  fuchsin.  The  cause  of  black-leg  in  cattle. 

III. — Bacillus  tetani,  sporulating;  pure  culture  in  glucose  bouillon, 
stained  with  fuchsin.  The  cause  of  lockjaw  in  the  horse. 

IV. — Bacillus  mallei,  pure  culture  on  glycerin  agar  ;  stained  with 
methylene  blue.  The  cause  of  glanders  in  the  horse. 

V. — Bacillus  of  tuberculosis  from  the  gland  of  a  hog.  Stained  with 
carbol-fuchsin. 

VI. — Spirillum  or  vibrio  of  Metchnikoff,  from  a  pure  culture  on  agar, 
stained  with  fuchsin.  The  cause  of  vibrio  cholera  in  the  chicken. 

VII. — Two  halteridia  infecting  the  red  blood  corpuscles  of  a  bird.  A 
protozoan  organism  and  the  cause  of  avian  malaria. 

VIII. — A  representative  of  the  flagellate  protozoan  organism  trypano- 
soma.  The  cause  of  surra,  nagana,  etc.,  in  horses,  cattle,  and  other 
domestic  animals. 


PLATE   I 


QUESTIONS  25 

animals.  It  derives  its  nutrition  from  the  contents  of  the  large  intes- 
tine, and  is  harmless  except  when  it  invades  such  organs  as  the  gall- 
bladder, the  urinary  bladder,  etc. 

Symbiotes. — There  are  some  parasitic  microorganisms  which  are  not 
only  harmless  but  which  are  beneficial  or  even  absolutely  necessary. 
For  instance,  there  are  bacteria  living  on  the  roots  of  higher  plants 
(clover)  without  which  these  plants  could  not  obtain  their  nitrogen 
supply.  Certain  bacteria  occur  in  the  human  and  animal  vagina 
which  keep  the  secretion  of  the  organ  acid  and  tend  to  prevent  dis- 
ease-producing microorganisms  from  gaining  entrance  into  the  uterus. 
Such  necessary  or  beneficial  parasites  are  called  symbiotes.  The  term 
symbiosis  is,  however,  also  used  in  bacteriology  in  a  very  different 
sense — namely,  for  two  bacteria  living  together  and  producing  by 
their  united  efforts  a  more  virulent  form  of  disease. 


QUESTIONS. 

1.  What  are  the  distinguishing  characters  between  an  animate  and  an  inan- 
imate object  of  nature? 

2.  What  is  an  organ?     What  an  organism? 

3.  What  is  a  unicellular  organism? 

4.  What  is  a  microorganism  or  microbe  ? 

5.  What  is  a  pathogenic  microorganism? 

6.  What  is  an  infectious  disease? 

7.  Name  some  diseases  which  occur  only  in  man  and  not  in  the  domestic 
animals  ? 

8.  Name  some  diseases  which  occur  in  domestic  animals  and  not  in  man. 

9.  Name  some  diseases  common  to  man  and  to  domestic  animals. 

10.  Name  a  harmless  non-pathogenic  microorganism  which  looks  very  much 
like  the  anthrax  bacillus. 

11.  What  is  the  role  of  the  microorganisms  in  general  in  nature  ?     Why  are 
they  essential  for  the  maintenance  of  life  on  our  planet? 

12.  What  is  the  meaning  of  the  term  saprophyte? 

13.  What  is  the  meaning  of  the  term  parasite? 

14.  What  does  the  term  ubiquitous  mean? 

15.  What  is  an  obligate  parasite?    Name  such  a  microorganism. 

16.  Is  the  anthrax  bacillus  an  obligate  parasite  or  not? 

17.  What  is  a  facultative,  what  an  obligate  saprophyte? 

18.  "V^hat  is  an  ectogenous  parasite? 

19.  What  is  an  entogenous  parasite? 

20.  What  is  a  commensale? 

21.  Name  a  bacillus  which  lives  in  the  intestines  of  man  and  domestic  animals 
as  a  commensale. 

22.  What  are  symbiotes? 

23.  What  does  the  term  symbiosis,  when  used  in  connection  with  pathogenic 
bacteria,  generally  signify  ? 


CHAPTEE    III. 

BACTERIA— GENERAL  CONSIDERATIONS— MORPHOLOGY. 

Definition. — Bacteria  (singular  bacterium)  are  very  minute,  uni- 
cellular, vegetable  microorganisms,  of  round,  cylindrical,  or  spiral 
shape,  motile  or  immotile,  which  perform  their  nutritive  function 
without  the  aid  of  chlorophyl,  and  which  multiply  very  rapidly  by 
binary  division  or  fission. 

Position  among  Organisms. — Bacteria  have  been  placed  as  an  inter- 
mediary phylum  between  animals  and  plants  by  some  biologists  and 
by  others  they  have  been  classified  as  the  lowest  forms  of  animal 
life,  because  many  are  motile.  If  we  consider  their  mode  of  nutri- 
tion without  the  aid  of  the  chlorophyl  of  the  higher  plants  and  their 
whole  metabolism  we  find  that  they  are  nearest  related  to  the  higher 
fungi.  Their  most  logical  classification  is,  therefore,  among  the  plants. 
On  account  of  their  mode  of  multiplication  by  division  they  are  also 
called  fission  fungi,  or  schizomycetes.  Bacteria  do  not,  like  the  higher 
plants,  show  any  differentiation  into  root,  stem,  or  leaves,  but  consist 
generally  of  a  very  simple  single  cell. 

Types. — When  bacteria  were  first  studied  by  botanists  and  biolo- 
gists in  general  (as,  for  instance,  Naegeli  and  Zopf)  it  was  believed 
that  they  were  very  variable  in  form,  and  that  one  and  the  same 
species  might  present  itself  alternately  in  ball-,  rod-,  or  screw-form. 
The  botanist  F.  Cohn  was  the  first  to  claim  that  this  was  an  erroneous 
impression,  and  that  bacteria,  like  higher  plants,  were  constant  in 
shape.  This  was  proved  beyond  doubt  by  Robert  Koch  and  his 
followers.  For  the  purpose  of  obtaining  so-called  pure  cultures  of  a 
single  species  of  bacteria,  Koch  for  the  first  time  devised  and  used 
solid  culture  media,  which  has  enabled  us  to  show  beyond  doubt  that 
bacteria  are  constant  in  form,  and  that  each  type  only  reproduces 
itself.  Three  main  types  of  bacteria  are  distinguished: 

1.  The  coccus  (plural  cocci). 

2.  The  bacillus  (plural  bacilli). 

3.  The  spirillum  (plural  spirilla). 

The  coccus  is  a  ball  or  spherical-shaped  bacterium.  The  bacillus  is  a 
cylindrical  rod-shaped  bacterium  very  much  like  a  short,  round  lead 
pencil.  The  spirillum  is  spiral  or  corkscrew-like  in  shape.  It  may 
consist  of  a  portion  of  a  screw  winding,  or  it  may  show  several  twists, 
giving  it  the  appearance  of  a  complete  corkscrew.  In  the  former 
case  we  speak  of  a  vibrio,  while  in  the  latter  we  designate  the  complete 
spiral  as  a  spirillum  (plural  spirilla),  or,  better  still,  as  a  spirochete 
(plural  spirochetci). 


DEVIATION 


27 


These  various  types  in  propagation  or  multiplication  always 
reproduce  their  own  type,  but  it  must  be  stated  that  under  varying 
conditions  bacteria  sometimes  vary  from  their  most  typical  shape. 
For  instance,  the  cause  of  pneumonia  in  man  and  animals  is  a  double 


FIG.  4 


O 


5) 


«  b          c  d 

Types  of  bacteria  (schematic):    a,  coccus;  6,  bacillus;  c,  vibrio;  d,  spirillum. 

coccus,  a  so-called  diplococcus,  which  in  artificial  cultures  sometimes 
becomes  elongated,  so  that  it  is  lancet-shaped  instead  of  ball-shaped. 
Other  cocci  may,  under  certain  conditions,  become  flattened,  so  that 
they  look  like  a  half-moon  or  somewhat  like  a  crescent.  Sometimes 
bacilli  in  multiplying  may  become  very  short,  so  that  they  look  on 


FIG.  5 


FIG. 


Very  large  spirilla.    (Park.) 


Medium-sized  spirilla. 


superficial  examination  like  cocci.  The  three  kinds  named,  however, 
never  change  their  shape  permanently  or  so  completely  that  they 
really  assume  another  type. 

Deviation  (Pleomorphous  Bacteria). — Some  bacteria,  however,  may 
show,  under  certain  conditions,  not  always  well  understood,  certain 
deviations  from  the  common  normal  or  average  type.  They  may,  for 


28    BACTERIA,  GENERAL  CONSIDERATIONS,  MORPHOLOGY 


FIG.  7 


Involution  forms  from  bacilli.    (From 
Fliigge.) 

FIG.  8 


instance,  form  true  branches.  The 
tubercle  bacillus,  the  glanders  bacillus, 
and  the  diphtheria  bacillus  belong  to 
that  type  of  bacteria  which  form  occa- 
sionally true  branches  commonly  found 
among  higher  microorganisms  of  a 
vegetable  type,  namely,  the  moulds. 
Bacteria  which  in  this  respect  deviate 
from  the  normal  or  average  type  are 
called  pleomorphous  bacteria.  Other 
bacteria,  as,  for  instance,  the  Bacillus 
proteus  vulgaris,  may  form  pseudo- 
branches,  that  is,  false  branches  due 
merely  to  an  arrangement  which  resem- 
bles the  preceding  type.  Such  pseudo- 

Fio.  9 


Glanders  bacillus.     (Wherry.) 
FIG.  10 


Bacillus  of  bubonic  plague.     (Herzog.) 
FIG.  11 


Bacillus  of  bubonic  plague.     (Herzog.)  Bacillus  of  bubonic  plague.     (Herzog.) 

Figs.  8  to  11  illustrate  various  types  of  involution  forms. 


INVOLUTION  FORMS 


29 


branches  are  formed  in  a  chain  of  bacilli  when  one  near  the  centre 
multiplies,  and  in  doing  so  pushes  the  new  bacillus  formed  out 
of  the  line,  so  that  it  projects  to  either  side,  but  still  retains  its 
connection  with  the  bacillus  which  produced  it. 

Involution  Forms.  —  Bacteria  often  show  very  abnormal  forms  under 
unfavorable  conditions  of  growth,  but  these  must  simply  be  looked 
upon  as  degenerates,  or  cripples.  They  are  known  as  involution 
forms.  For  example,  the  coccus  of  pneumonia  on  artificial  culture 
media  shows  long,  irregular,  bacilli-like  forms;  the  anthrax  bacillus, 
which  is  a  stiff,  straight,  cylindrical  rod,  becomes  curved;  the  plague 
bacillus  in  old  cultures  forms  spermatozoa-like  bodies,  and  the  same 
bacillus  on  an  artificial  culture  medium  (agar),  containing  3  to 
4  per  cent,  of  common  salt,  forms  large,  round,  yeast,  cell-like 
balls.  In  very  old  cultures  bacteria  undergo  splitting  up  or  frag- 

FIG.  12 


a          b  c  d  e  f  g  h  i 

Bacillus  Biitschli:  a  to  c,  incomplete  division  of  the  cell;  d  to  /,  gradual  collection  of  chro- 
matin  granules  at  ends  of  cells;  g  to  i,  formation  of  end  spores  from  these  chromatin  end-masses. 
(After  Schaudinn.) 

mentation,  and  may  finally  break  up  into  irregular  granules.  How- 
ever, as  soon  as  such  involution  forms  are  placed  in  fresh,  good 
culture  media,  under  favorable  conditions,  they  assume  again  their 
normal  shape,  with  all  of  their  normal  average  properties.  It  is 
only  when  unfavorable  conditions  are  present,  such  as  exhaustion  of 
the  soils,  accumulation  of  metabolic  products,  etc.,  that  the  foregoing 
changes  take  place,  otherwise  bacteria  always  reproduce  their  own 
type  and  are  not  polymorphous.  Involution  forms  are,  as  a  rule, 
live  bacteria,  and  if  they  are  pathogenic  they  can  produce  their 
specific  disease.  If  inoculated  into  a  fresh  culture  soil  they  will 
reproduce  the  normal  type  of  the  species. 

Bacteria  can  be  killed  with  chloroform  or  formalin  vapors  in  such 
a  manner  that  they  retain  their  shape  perfectly.  This  should  be  done 
when  cultures  of  dangerous  bacteria  are  placed  in  the  hands  of  begin- 
ners in  the  laboratory  study  of  pathogenic  microorganisms. 


30      BACTERIA,  GENERAL  CONSIDERATIONS,  MORPHOLOGY 


FIG.  13 


r :- 


Cell  Structure. — The  cell  which  forms  the  body  of  a  bacterium  does 
not  show  any  well-marked  differentiation  into  a  protoplasmic  body 
and  a  nucleus.  Most  of  the  substance  of  the  bacterium,  however, 
takes  the  so-called  nuclear  stains,  particularly  the  basic  anilin  stains, 
and  extensive  studies  have  shown  that  most  of  the  body  consists  of 
diffusely  distributed  nuclear  substance  called  chromatin.  The  latter 
is  not  contained  in  a  well-defined  nuclear  membrane,  as  in  cells  of 
higher  plants  and  animals,  but  is  intimately  mixed  with  a  scanty 
amount  of  protoplasm  called  the  entoplasm,  around  which  there  is  often 
a  very  small  rim  of  ectoplasm.  In  certain  very  young  bacteria,  or  in 
bacteria  which  are  at  rest  and  not  dividing  a  small  nucleus-like  body, 

can  sometimes  be  demonstrated, 
but  in  all  actively  dividing  bac- 
teria the  chromatin  fills  the 
interior  of  the  cell  and  is  present 
to  such  an  extent  that  it  almost 
completely  hides  the  scanty 
amount  of  entoplasm. 

Bacteria  often  contain  distinct 
granules  which  in  the  unstained 
condition  are  highly  refractive, 

\  /       and  when  stained  take  the  dye 

in     a    very     intense     manner. 

^JtjjJL     m  These   granules   are  known   as 

the  metachromatic  bodies  or  the 
polar  bodies,  since  they  are  found 
at  one  or  both  ends  of  a  bacillus. 
They  are  also  called  the  Babes- 
Ernst  granules,  after  the  two  in- 
vestigators who   first  described 
them    minutely.      These    polar 
bodies  must  not  be  confounded  with  the  sporogenous  granules  (see 
below). 

The  ectoplasm  of  the  bacteria  does  not  usually  stain  by  the 
ordinary  methods  used.  While  generally  scanty,  the  ectoplasm  may 
be  more  powerful,  particularly  in  bacteria  with  many  flagella  (see 
below).  It  is  believed,  and  perhaps  fairly  well  demonstrated, 
that  bacteria  generally  possess  a  membrane  between  the  ectoplasm 
and  the  endoplasm,  and,  as  a  rule,  some,  under  definite  conditions, 
possess  outside  of  the  ectoplasm  a  smaller  or  larger  gelatinous 
capsule  surrounding  the  bacteria.  The  jelly-like  masses  forming 
these  capsules  may  become  confluent,  and  so  form  one  gelatinous 
matrix  in  which  the  bacteria  are  embedded  like  cells  of  higher 
animals  in  an  intercellular  substance.  Such  formations  are  known 
as  zoogloea  or  zoogloeal  masses. 

Flagella. — If  we  study  various  bacteria  in  the  live  state  in  a  drop 
of  water  or  other  suitable  fluid  we  will  notice  some  that  possess  the 


Postmortem  smear  from  the  heart  blood 
in  a  case  of  bubonic  plague,  showing  one 
plague  bacillus  in  the  centre  of  the  field,  with 
polar  bodies  stained  very  deeply.  X  2000. 
(Author's  preparation.) 


BROWNIAN  MOVEMENT 


31 


power  of  locomotion.  These  motile  bacteria,  when  observed  in  fluid, 
shoot  and  dart  about  like  a  school  of  minnows  in  clear  water.  Bac- 
teria which  are  truly  motile  possess  organs  of  locomotion  in  the 
shape  of  exceedingly  fine  slender  threads  or  filaments  called  flagella 
(singular  flagellum).  These  filaments  are  found  at  one  or  both  ends, 
or  all  around  the  body.  Generally,  only  bacilli  and  spirilla  possess 
flagella,  but  a  very  few  cocci  also  have  them.  We  classify  flagellate 
bacteria  as  follows: 

Monotricha — one  flagellum  at  one  end. 

Amphitricha — one  flagellum  at  each  end. 

Lophotricha — several  flagella  at  one  end. 

Peritricha — flagella  all  around  the  bacterium. 

Atricha — no  flagella. 


FIG.  14 


FIG.  15 


Fraenkel's  pneumococcus,  pure  culture  in 
litmus  milk,  showing  capsule.  X  1000. 
(Author's  preparation.) 


Anthrax  bacillus  in  the  blood  of  an  infected 
cow,  showing  capsule.  X  1000.  (From  a 
preparation  of  Dr.  L.  E.  Day.) 


Characteristics. — Flagella  are,  as  a  rule,  exceedingly  slender  fila- 
ments, several  times  longer  than  the  bacterium  itself.  They  are  wavy 
in  outline  and  terminate  in  a  blunt  or  slightly  club-shaped  extremity. 
They  cannot  be  stained  by  the  ordinary  bacterial  staining  methods, 
but  require  a  special  complicated  technic.  They  break  off  easily 
from  the  bacterium,  but  as  far  as  known  they  are  easily  regenerated. 
They  arise  out  of  the  ectoplasm,  but  are  probably  also  connected 
with  the  entoplasm. 

Brownian  Movement. — Even  those  bacteria  which  have  no  flagella, 
and  which  in  consequence  have  no  true  locomotion  when  examined 
in  a  drop  of  water,  present  a  peculiar  oscillating,  trembling  motion, 
but  this  movement  is  not  confined  to  living  bacteria.  Dead  bacteria 
or  very  minute  particles  of  solid  matter,  when  suspended  in  fluid, 
show  a  similar  motion,  which  is  called  the  Brownian  movement, 
because  it  was  first  described  by  the  botanist  Brown.  It  is  now 


32      BACTERIA,  GENERAL  CONSIDERATIONS,  MORPHOLOGY 


known  that  this  motion  of  very  small  solid  particles  in  fluid  is  due 
to  the  fact  that  the  adjacent  particles  of  gaseous  or  liquid  matter 
surrounding  them  are  in  constant  violent  motion,  and  these  lively 


FIG.  16 


FIG.  17 


Spirillum  of  Asiatic  cholera,  showing  single 
flagellum.     (Kolle  and  Zetnow.) 


Spirillum  volutans,  showing  flagella  at 
either  end  of  the  bacterium. 


molecules  bumping  constantly  against  the  small,  not  truly  motile 
bacteria  keep  them  in  a  continued  state  of  trembling  agitation.  It 
was  formerly  believed  that  the  Brownian  movement  depended  upon 
surface  tension,  but  this  is  not  the  case. 

Multiplication    or  Propagation. 

—Under 

bacteria 

material 


FIG.  18 


suitable  conditions 
take  up  nutritive 
and  multiply  very 
quickly.  This  process  is  so 
rapid  that  a  single  bacterium, 
if  circumstances  are  favorable, 
may  in  twenty-four  hours  have 
increased  to  many  millions.  It 
has  been  ascertained  that  several 
of  the  disease-producing  bac- 
teria under  the  most  favorable 
conditions  divide  once  in  less 
than  thirty  minutes. 

Binary  Division  or  Fission. — 
It  is  characteristic  of  bacteria 
that  they  always  multiply  by 
dividing  in  the  middle  and  the 
elongated  forms — namely,  bacilli 
and  spirilla  always  divide  at  right  angles  to  their  long  axis.  This 
mode  of  multiplication  is  called  binary  division,  or  fission,  and  often 
causes  bacteria  to  group  themselves  in  a  very  characteristic  manner, 


Bacillus  proteus  vulgaris,  showing  numerous 
fla^ella  around  the  entire  body  of  the  bac- 
terium. 


SHAPE  AND  ARRANGEMENT  OF  &ACILLI  33 

which  has   given   rise   to    a   subdivision  of   the   cocci  into  several 
groups. 

Subdivision  of  Cocci. — Cocci,  after  the  first  division,  may  adhere  to 
each  other,  but  after  the  second  division  usually  separate,  generally 
forming  in  groups  of  two,  called  diplococci.  They  may,  however, 
adhere  together  until  after  the  second  division,  in  which  case  they 
form  groups  of  four,  called  tetrads  (tetra — Greek  word  for  four). 
Again  they  may  divide  in  all  three  directions  of  space  and  adhere 
together,  then  we  obtain  square  packages  of  cocci  called  sarcina. 
Yet,  again,  division  may  be  in  one  direction  only,  when  there  are 
formed  regular  rows  or  chains  or  cocci,  called  streptococci  (chain- 
cocci).  Finally,  the  division  of  the  cocci  may  go  on  in  an  irregular 
manner  in  all  three  dimensions  of  space,  resulting  in  the  formation 
of  irregular  clusters,  resembling  bunches  of  grapes,  which  are  called 
staphylococci.  f 

FIG.  19 


Varieties  of  spherical  forms:  a,  tendency  to  lancet-shape;  6,  tendency  to  coffee-bean   shape; 
c,  in  packets;  d,  in  tetrads;  e,  in  chains;  f,  in  irregular  masses.     X  1000.     (After  Flugge.) 

Classification  of  Cocci. — The  single  coccus,  or  micrococcus. 

The  diplococcus,  or  group  of  two  cocci. 

The  tetrad,  or  group  of  four  cocci. 

The  sarcina,  or  cuboidal  (dice-like)  group  of  eight  or  sixteen  or 
more  cocci. 

The  staphylococci,  or  several  cocci  irregularly  arranged  like  a 
bunch  of  grapes. 

The  streptococci,  or  a  group  of  several  cocci  arranged  like  a  row  of 
beads  (chain  cocci). 

Shape  and  Arrangement  of  Bacilli. — Bacilli  shows  varying  features 
as  to  details  of  shape.  Some  are  quite  slender,  others  plump,  thick, 
and  short.  Some  of  them,  like  the  anthrax  bacillus,  are  cylindrical, 
straight,  and  stiff,  with  square  ends,  while  others  show  rounded  ends, 
like  the  typhoid,  colon,  and  hog-cholera  bacilli.  There  are  bacilli 
which  are  pointed  at  one  end  and  club-shaped  at  the  other,  or  club- 
shaped  at  both  ends,  like  the  diphtheria  and  glanders  bacilli.  Again, 
others,  instead  of  being  straight,  are  often  slightly  but  distinctly 
curved  like  the  tubercle  bacillus.  Certain  bacilli,  as  the  anthrax, 
3 


34      BACTERIA,  GENERAL  CONSIDERATIONS,  MORPHOLOGY 

typhoid,  colon,  and  hog  cholera,  have  a  tendency  to  form  long  chains; 
others,  like  the  tetanus,  group  themselves  in  short  chains  of  two  or 


FIG.  20 


Streptococcus  in  pus,  showing  how  the  chain  is  formed  by  groups  of  two  (diplococci). 
The  cells  seen  in  the  field  are  polynuclear  leukocytes,  with  the  exception  of  one  cell,  which  is 
a  mononuclear  leukocyte.  X  1000.  (Author's  preparation.) 

three.  Still  other  bacilli  form  long  chains  in  which  the  lines  of 
division  between  the  individual  bacilli  cannot  be  seen,  so  that  we 
see  pseudofilaments.  Certain  bacilli  rarely  if  ever  form  chains,  but 

FIG.  21 


Various  forms  of  bacilli:  a,  bacilli  with  sides  parallel  to  their  long  axis  and  with  ends 
perpendicular;  6,  bacilli  with  sides  swollen  or  narrowed,  causing  irregular  forms.  X  1000. 
(After  Fliigge.) 

generally  fall  apart  after  division  and  form  parallel  groups.    When 
bacilli  are  studied  in  stained  preparations  some  appear  very  uni^ 


SPORULATION   OR  SPORE  FORMATION  35 

formly  colored,  others  stain  in  such  a  manner  that  dyed  portions 
alternate  with  undyed  sections  of  the  entoplasm.  Some  bacilli  take 
the  stains  easy,  others  with  difficulty. 

Size  of  Bacteria. — Microscopic  Measure. — Bacteria  are,  as  a  rule, 
exceedingly  small  in  size,  their  longest  diameter  being  only  a  fraction 
of  the  diameter  of  a  mammalian  red  blood  corpuscle,  and  they  can  be 
studied  individually  only  by  the  aid  of  good  compound  microscopes. 

The  size  of  bacteria  is  expressed  by  a  microscopic  measure  based 
upon  the  metric  system: 

1  meter  (about  forty  inches)  =  100  centimeters  =  1000  millimeters. 

1  millimeter  =  1000  micromillimeters. 

1  micromillimeter  or  micron  =  about  2~5iroir  incn- 

The  term  micromillimeter  is  indicated  by  the  Greek  letter  p  or 
abbreviated  micron  (plural  micra). 

Sporulation  or  Spore  Formation. — Under  certain  conditions,  par- 
ticularly when  the  soil  in  which  bacteria  have  grown  abundantly 
becomes  exhausted,  and  when  the  metabolic  products  have  accumu- 
lated, spore  formation  occurs.  The  spore  of  a  bacterium  may  be 
likened  in  a  certain  sense  to  the  seed  of  a  higher  plant.  Only  a 
limited  number  of  the  disease-producing  bacteria  form  spores,  and 
these  are  nearly  all  bacilli,  very  rarely  cocci  and  spirilla.  Spore 
formation  is  sometimes  dependent  upon  very  definite  conditions,  for 
instance,  the  anthrax  bacillus  requires  the  presence  of  free  oxygen. 
Because  these  spores  are  formed  in  the  interior  of  the  bacterium 
they  are  known  as  endospores.  Bacteria  do  not  multiply  by  sporu- 
lation,  since  there  is  only  one  spore  formed.  However,  this  is  not 
an  absolute  rule;  exceptionally  two  spores  have  been  found  in  one 
bacillus,  but  this  occurrence  is  so  very  rare  that  it  may  be  neglected 
entirely  for  any  practical  consideration.  Spore  formation  is  very 
important  in  the  life  history  of  bacteria,  because  the  spores  are  very 
resistant  to  external  inimical  influences,  and  can  stand  antiseptics 
and  heat  very  much  better  than  the  full-grown  or  vegetative  form 
of  the  bacterium.  In  fact,  some  spores,  as,  for  instance,  those  of  the 
bacilli  of  tetanus,  malignant  edema,  black-leg,  and  some  soil  bac- 
teria, represent  the  most  resistant  organisms  known.  In  order  to 
kill  tetanus  spores  with  certainty  they  must  be  exposed  for  over  an 
hour  to  the  temperature  of  boiling  water  or  steam  at  100°  C.  This 
great  resistance  enables  spores  to  survive  where  the  adult  vegetative 
form  of  the  bacterium  would  perish.  On  account  of  their  great 
resistance,  spores  in  German  are  known  as  Dauerformen,  which 
means  durable  forms  of  the  bacterium.  The  great  resistance  of 
spores  is  largely  due  to  the  fact  that  they  possess  a  very  firm,  tough, 
protecting  membrane.  In  shape  they  are  either  round  or  oval,  and 
are  situated  at  either  the  centre  or  at  or  near  one  of  the  ends  of  the 
bacterium.  Spores  situated  at  one  end  of  the  bacterium,  like  that  of 
the  tetanus  bacillus,  give  to  the  small  rod  the  appearance  of  a  drum- 
stick. Spores  situated  in  the  centre,  and  making  this  part  bulge  out, 


36      BACTERIA,  GENERAL  CONSIDERATIONS,  MORPHOLOGY 

give  the  bacillus  a  somewhat  barrel-shaped  appearance.     Such  a 
bacillus  is  known  as  a  clostridium. 

Sporogenous  Granules. — When  spore  formation  is'  about  to  occur 
in  a  bacterium  there  appears  in  its  interior,  first,  a  dust-like  trans- 
formation of  the  protoplasm,  which  gives  it  a  powdered  appearance; 
next  appears  one  or  more  highly  refractive  bodies,  the  so-called  sporog- 
enous  granules.  These  become  confluent,  and  from  them  the  spore 
is  formed  as  a  highly  refractive  body,  composed  of  condensed  proto- 
plasm, and  surrounded  by  a  very  firm,  tenacious,  tough  membrane. 
The  spore  may  escape  from  the  bacillus  at  one  end  or  it  may  rupture 
the  bacterium  in  the  equatorial  plane.  When  a  spore  is  placed  under 
favorable  conditions  it  takes  up  food  material,  its  capsule  ruptures, 

FIG.  22 


Bacillus  subtilis  sporulating.     The  unstained  spaces  in  the  centre  of  the  rod  are  spores. 
(Author's  preparation.) 

and  from  its  protoplasm  is  formed  the  ordinary,  typical,  vegetative 
variety  of  the  bacterium  from  which  the  spore  was  originally  formed. 
This  process  of  the  formation  of  the  adult  vegetative  form  of  the 
bacterium  from  its  spore  is  called  the  germination  of  the  spore. 

Arthrospores. — Cocci  sometimes  appear  to  change  their  whole  body 
into  a  spore.  These  supposed  spores  were  called  arthrospores. 
Our  knowledge  of  this  type  is  limited  and  requires  further  study, 
but  it  is  thought  that  the  so-called  arthrospores  are  merely  invo- 
lution forms.  Of  the  disease-producing  bacteria  we  may  name  as 
examples  of  spore-formers  the  bacilli  of  anthrax,  tetanus,  malignant 
edema,  emphysematous  anthrax,  or  black-leg;  of  harmless  sapro- 
phytes the  Bacillus  subtilis  and  the  Bacillus  megatherium.  Spores 
cannot  be  stained  by  the  ordinary  methods  used  to  dye  the  vegetative 
forms. 


QUESTIONS  37 


QUESTIONS. 

1.  What  unicellular  vegetable  microorganisms  are  classified  as  bacteria? 

2.  Why  are  bacteria  best  classified  as  plants? 

3.  Why  are  they  called  fission  fungi,  or  schizomycetes  ? 

4.  Do  bacteria  in  the  course  of  their  multiplication  change  their  shape  ? 

5.  What  are  the  three  main  morphologic  types  of  bacteria?    Name  and 
describe  them. 

6.  What  method  has  enabled  us  to  establish  the  constancy  of  the  morpho- 
logic features  of  bacteria  ?    Who  devised  this  method  ? 

7.  What  is  the  difference  between  a  vibrio  and  a  spirochete  ? 

8.  Do  bacteria  in  their  multiplication  under  variable  conditions  vary  at  all, 
and  if  so  under  what  conditions? 

9.  What  is  meant  by  pleomorphous  bacteria  ?    Describe  their  characteristics 
and  name  some. 

10.  What  is  meant  by  a  pseudobranching  of  multiplying  bacteria?    Name  an 
example. 

11.  What  are  involution  forms  of  bacteria?    Under  what  conditions  are  they 
formed  ?    Give  some  examples. 

12.  Are  all  involution  forms  dead  organisms  ?    What  becomes  of  the  involution 
forms  when  they  are  inoculated  into  a  fresh  culture  soil  ? 

13.  How  are  bacteria  killed  so  that  they  retain  their  normal  morphologic 
features  without  degenerating  into  involution  forms  f 

14.  Describe  the  finer  structure  of  the  bacterial  cell. 

15.  How  is  the  chromatic  substance  of  the  bacterium  arranged? 

16.  What  is  the  entoplasm,  the  ectoplasm,  the  membrane  of  a  bacterium? 

17.  What  is  meant  by  the  metachromatic  granules  of  a  bacterium?    By  what 
other  names  are  they  also  designated? 

18.  What  is  meant  by  the  gelatinous  capsule  of  a  bacterium?    What  by  a 
zodgloa  or  a  zoogloal  mass? 

19.  What  is  the  difference  between  a  motile  and  a  non-motile  bacterium  ?    Have 
the  former  any  organs  of  locomotion?    How  called?    Describe  in  detail  these 
organs  of  locomotion. 

20.  How  are  bacteria  classified  according  to  the  number  and  arrangement 
of  their  flagella. 

21.  What  types  of  bacteria  generally  possess  flagella? 

22.  What  is  meant  by  the  Brownian  movement  of  a  bacterium?    What  is  it 
due  to? 

23.  Name  and  describe  the  various  types  of  cocci  which  result  from  the  various 
modes  of  fission  which  occur  in  these  ball-shaped  bacteria. 

24.  Name  and  describe  various  morphologic  types  of  bacilli. 

25.  What  is  a  streptobacillus? 

26.  What  are  pseudofilaments ? 

27.  What  measure  is  employed  to  express  the  size  of  bacteria?    What  is  the 
meaning  of  the  Greek  letter  ^  in  bacteriologic  nomenclature  ?    What  is  the  mean- 
ing of  the  terms  micron  and  micra? 

28.  What  is  a  bacterial  spore?    Describe  its  appearance. 

29.  Why  is  a  bacterial  spore  called  entogenous  ? 

30.  Does  spore  formation  occur  in  all  bacteria  and  under  all  conditions?     If 
not,  in  what  kind  of  bacteria  does  it  occur,  and  when  ? 

31.  Name  some  disease-producing,  spore-forming  bacteria. 

32.  How  many  spores  does  a  bacterium  form  ? 

33.  What  occurs  in   a   bacterium    during   spore  formation?     Describe    the 
phenomena  in  detail.    Give  location  of  spores  in  bacilli. 

34.  What  is  a  clostridium  ? 

35.  Why  is  a  spore  called  a  "Dauerform"  (durable  form  of  the  bacterium)? 

36.  What  is  meant  by  the  vegetative  form  of  a  bacterium  ? 

37.  How  and  when  do  spores  change  into  the  vegetative  forms  of  their  respec- 
tive species. 

38.  What  is  meant  by  sporulation ?    What  by  germination? 


CHAPTEE    IV. 

BIOLOGY  OF  BACTERIA. 

IN  the  previous  chapter  the  morphology  of  bacteria  has  been  con- 
sidered. It  is,  however,  impossible  strictly  to  separate  the  mor- 
phology from  the  biology  of  these  organisms.  Involution  forms  and 
spores  are  certainly  morphologic  features  of  bacteria,  yet  they  cannot 
be  referred  to  without  going  to  some  extent  into  the  biology  of  the 
fission  fungi.  The  present  chapter  will  be  more  particularly  devoted 
to  some  important  features  in  the  life  history  of  bacteria,  as  they 
are  dependent  upon  varying  conditions  of  their  existence  with  refer- 
ence to  environments,  nutrition,  metabolism,  etc.  It  has  previously 
been  stated  that  bacteria  are  as  a  class  ubiquitous,  that  is,  they  are 
found  everywhere  on  or  near  the  surface  of  our  globe,  in  soil,  water, 
air,  or  the  external  surface  of  the  bodies  of  plants  and  animals,  and 
in  the  intestinal  tract  of  the  latter.  They  are  not  found  in  the  blood 
or  interior  of  tissues  of  healthy  animals,  nor  to  any  extent  in  the 
highest  altitudes  and  latitudes. 

Temperature  Limits. — A  most  remarkable  feature  of  bacterial  life 
in  general  is  that  they  as  a  class  can  exist  and  multiply  under  a  wider 
range  of  temperature  than  any  other  class  of  organisms.  There  are 
bacteria  that  multiply  in  sea  water  at  0°  C.  (32°  F.)  and  others 
that  multiply  in  springs  at  75°  C.  (167°  F.).  Many  individual  species 
exist  and  multiply  under  a  wide  range  of  temperature,  but  some, 
particularly  the  pathogenic  strict  parasites  of  warm-blooded  animals, 
are  quite  limited  in  the  latitude  of  temperature  under  which  they 
can  exist  and  grow.  Bacteria,  as  a  rule,  flourish  and  multiply  most 
rapidly  at  a  definite  temperature  called  their  optimum  temperature. 
Beyond  certain  limits  above  and  below  the  optimum  temperature 
they  will  not  grow  at  all;  these  limits  are  called  the  maximum  and 
minimum  temperature  of  their  growth.  The  bacillus  of  mam- 
malian tuberculosis  has  its  optimum  temperature  at  37°  to  38°  C., 
that  of  avian  tuberculosis  at  38°  to  43°  C.;  the  former  its  minimum 
temperature  at  29°  C.,  the  latter  at  35°  C.;  while  their  maximum 
temperatures  are  41°  C.  and  46°  C.,  respectively.  These  are  examples 
of  strictly  parasitic  bacilli  which  have  a  very  narrow  range  of  tem- 
perature at  which  they  can  grow  and  multiply.  This  is  also  true  of 
the  strict  saprophytes.  On  the  other  hand,  facultative  parasites,  which 
occur  also  as  saprophytes,  often  have  the  wide  range  of  25°  C.  from 
15°  to  40°  C.,  and  more. 

Thermophile  Microorganisms. — Bacteria  which  multiply  best  at 
very  high  temperatures  (75°  C.)  are  called  thermophile,  which,  literally 


NUTRITION  OF  BACTERIA  39 

translated,  means  heat-loving.  They  probably  first  appeared  on  our 
planet  at  a  time  when  its  surface  was  considerably  warmer  than  it  is 
now.  They  rarely  multiply  at  temperatures  below  40°  to  50°  C.,  and 
are  found  in  abundance  only  in  the  tropics,  in  hot  springs,  and  in  soil 
which  has  been  exposed  to  the  direct  rays  of  the  sun  for  some  time. 
They  are  quite  common  in  the  intestinal  contents  of  animals. 

Thermophile  bacteria  probably  multiply  considerably  in  sponta- 
neously fermenting  manure,  and  assist  in  bringing  about  by  their 
metabolism  the  marked  elevation  of  temperature.  Some  thermophile 
bacteria,  in  the  absence  of  free  oxygen,  may  be  able  to  multiply  at 
temperatures  as  low  as  34°  C.  Such  bacteria  may  possibly  multiply 
in  the  intestines  of  man  and  domestic  animals. 

Thermotolerant  Microorganisms. — There  are  some  bacteria  which 
multiply  at  quite  high  temperatures,  but  which  have  their  optimum 
at  35°  to  37°  C.  These  are  called  thermotolerant  (heat- tolerating).  Bac- 
teria in  their  vegetative  form,  with  the  exception  of  the  thermophile 
and  thermotolerant,  are,  as  a  rule,  not  very  resistant  to  heat.  They 
are  generally  killed  at  55°  to  60°  C.  if  exposed  in  the  moist  state  to 
this  temperature  for  about  ten  minutes.  The  exact  temperature 
and  time  has  to  be  ascertained  experimentally  for  each  species,  and 
again  for  the  spores  of  such  species  as  sporulate.  This  temperature 
when  applied  for  a  few  minutes  (generally  five  or  ten)  is  called  the 
thermal  death  point  of  the  bacterium  or  of  its  spore.  While  heat  easily 
damages  the  vegetative  form  of  the  bacteria,  cold,  as  a  rule,  has  very 
little  effect  upon  bacteria  and  their  spores.  They  may  be  frozen  at  a 
very  low  temperature  without  any  effect,  and  will  be  found  alive  after 
thawing.  However,  repeated  freezing  and  thawing  in  rapid  succes- 
sion kills  some  pathogenic  bacteria. 

Nutrition  of  Bacteria. — All  bacteria  depend  for  their  existence  and 
multiplication  upon  certain  food  materials  and  moisture.  Many  of 
them  may  be  dried  out  completely  (^dthooit-Jbeujg_Mlle(^,  but  they 
(cannot  multiply)  under  such  conditions,  and  only  do  so  after  they 
have  again  had  access  to  moisture.  Other  bacteria  when  dried  die 
very  soon,  as,  for  instance,  the  glanders  and  the  plague  bacilli.  Spores 
of  certain  pathogenic  bacteria,  like  those  of  anthrax,  tetanus,  black- 
leg, etc.,  can  exist  for  years  in  a  dried  condition,  and  when  conditions 
become  favorable,  germinate  and  display  all  of  their  general  typical 
and  special  pathogenic  properties.  Some  bacteria  can  exist  for  a 
long  time  in  their  vegetative  form  in  a  desiccated  state,  as,  for  instance, 
the  tubercle  bacillus.  Desiccated  bacteria  and  their  spores  must 
be  considered  as  being  in  a  condition  where  there  is  no  metabolism 
and  where  life  is  latent,  somewhat  like  life  in  higher  plants  in  winter 
or  in  hibernating  animals. 

When  bacteria  grow  in  artificial^  cultures  they  exhaust  the  soil 
within  a  certain  time,  which,  together  with  the  accumulation  of  their 
metabolic  products,  produce  conditions  destructive  to  most  of  them. 
They  die  sooner  if  they  are  raised  in  the  incubator  than  if  raised  at 


40  BIOLOGY  OF  BACTERIA 

room  temperature.  Bacteria  in  cultures  may  be  kept  alive  much 
longer  if  they  are  tightly  sealed  up  and  placed  in  the  refrigerator 
after  the  growth  has  been  well  developed.  This  not  only  keeps  them 
alive  much  longer,  but  prevents,  to  a  large  extent,  the  appearance  of 
involution  forms. 

Elements  Necessary  for  Growth. — Bacteria  need  for  their  nutrition, 
growth,  and  multiplication  substances  containing  the  elements 
carbon,  nitrogen,  hydrogen,  oxygen,  sulphur,  and  phosphorus,  and 
some  salts.  They  can  generally  best  derive  their  nutrition  from 
albumins  and  their  derivatives,  like  'peptones  and  gelatins,  but 
even  pathogenic  bacteria  may  be  grown  in  albumin-free  culture  soils 
composed  of  very  simple  compounds.  On  the  other  hand,  certain 
strictly  parasitic  pathogenic  bacteria  do  not  thrive  well  on  artificial 
culture  media,  particularly  during  the  first  generations.  The  bovine 
tubercle  bacillus  is  one  of  this  type.  Other  bacteria  have  never 
been  successfully  cultivated,  as,  for  instance,  the  leprosy  bacillus  or 
the  acid-fast  bacillus,  which  is  found  in  such  enormous  numbers  in 
Johne's  disease  of  cattle.  Other  pathogenic  bacteria  grow  only  after 
the  addition  of  unaltered  hemogoblin  to  the  culture  soil,  for  example, 
the  influenza  bacillus.  Still  others  require  the  addition  of  natural 
serous  exudates,  such  as  pleuritic  or  ascitic  fluid.  Too  great  a  con- 
centration of  the  culture  soil  with  a  high  percentage  of  solids  results 
in  a  poor  growth  or  prevents  it  entirely. 

Reaction  of  the  Medium. — All  bacteria  are  rather  particular  about  the 
reaction  of  the  medium  in  which  they  grow.  Some  favor  a  neutral, 
others  a  slightly  alkaline,  still  others  a  slightly  acid  medium.  Care 
must  be  taken  in  preparing  the  medium,  as  bacteria  generally  will 
not  thrive  if  it  is  more  than  slightly  acid  or  alkaline,  and  if  there 
is  any  marked  degree  of  either  the  bacteria  will  die,  especially  in 
the  presence  of  mineral  acids. 

Plasmolysis  and  Plasmoptysis. — Many  bacteria  cannot  stand  the 
sudden  transfer  from  one  fluid  to  another,  differing  materially  in 
concentration.  Such  sudden  changes  of  osmotic  pressure  may  cause 
a  shrinking  of  the  contents  of  the  bacterial  cell  away  from  the  mem- 
brane with  a  loss  of  fluid  to  the  outside  liquid;  this  is  called  plasmo- 
lysis.  Or  some  of  the  cell  contents  may  become  expelled  through  a 
rupture  in  the  membrane;  this  is  known  as  plasmoptysis.  These 
damaged  bacteria  are,  however,  not  necessarily  dead,  and  they  may 
return  to  the  normal  if  placed'again  under  favorable  conditions. 

Aerobic  and  Anaerobic  Bacteria. — Certain  bacteria  like  the  higher 
plants  and  animals  can  only  exist  and  grow  in  the  presence  of  free 
oxygen.  Such  bacteria  are  called  obligate  or  strict  aerobes.  To  this 
type  belong  many  saprophytes,  and  among  them  particularly  the 
color  or  pigment-forming  so-called  chromogenic  bacteria,  also  some 
microorganisms  which  form  poisonous  decomposition  products  in 
milk.  Among  the  pathogenic  bacteria,  the  plague  bacillus,  the 
influenza  bacillus,  the  pneumococcus,  etc.,  belong  to  this  group. 


METABOLIC  PRODUCTS  41 

The  opposite  type  of  bacteria  is  represented  by  the  obligate  or 
strict  anaerobes.  These  cannot  exist  and  multiply  in  the  presence  of 
free  oxygen,  hence  when  raised  in  artificial  cultures  the  air  must 
be  excluded  or  at  least  its  oxygen.  The  pure  nitrogen  present  does 
not,  as  a  rule,  interfere  with  the  growth  of  the  bacteria.  Among 
the  pathogenic  bacteria  the  most  important  obligate  or  strict  anae- 
robics  are  the  bacilli  of  tetanus,  black-leg,  malignant  edema,  and 
the  Bacillus  necrophorus.  Although  strictly  anaerobic  bacteria  die 
sooner  or  later  in  the  presence  of  free  oxygen,  their  spores  may  be 
exposed  a  long  time  before  being  killed.  Strict  anaerobes  may,  how- 
ever, exist,  grow,  and  multiply  in  the  presence  of  free  oxygen  when  they 
are  intimately  associated  with  aerobic  bacteria,  which  in  their  growth 
use  up  and  remove  the  oxygen  present. 

Facultative  aerobes  or  anaerobes  are  those  bacteria  which  can 
thrive  in  the  presence  or  absence  of  free  oxygen.  To  these  belong 
many  saprophytes  and  most  pathogenic  bacteria,  such  as  the  organ- 
isms of  anthrax,  of  typhoid,  of  cholera,  the  pus-producing  cocci,  etc. 

FIG.  23 


-  c 


Structure     of     bacterial  Plasmolysis:  a,  spirillum  undula;  6,  bacillus  solmsii;  c,  vibrio 

cell:     a,      endoplasm;     6,  choleras.     (After  A.  Fischer.) 

ectoplasm,  or  cell  mem- 
brane; c,  central,  less  in- 
tensely staining  parts;  d, 
chromatin  granules.  (After 
Butschli.) 

Metabolic  Products.  —  Bacteria  in  their  growth  and  metabolism  excrete 
certain  gaseous  and  soluble  waste  products:  among  them  are  carbon 
dioxide  (CO2),  volatile  compounds  of  nitrogen,  such  as  NH3,  also  free 
nitrogen,  hydrogen  sulphide  (H2S),  etc.  The  latter  is  formed  in  all 
putrefactive  processes  depending  uponbacteria  intheabsence  of  oxygen. 
Some  bacteria  in  the  presence  of  peptone  form  indol,  for  instance 
the  colon  bacillus,  and  this  product  may  be  used  in  differentiating 
nearly  related  bacteria  from  each  other  (the  typhoid  from  the  colon 
bacillus).  Indol  may  be  formed  in  consequence  of  putrefactive 
processes  or  by  chemical  decompositions  of  a  different  type.  Other 
chemical  reactions  brought  about  by  pathogenic  bacteria  are  the 
reduction  of  reducible  substances  with  oxygen  absorption  on  the  part 
of  the  bacteria,  reduction  of  nitrates  to  nitrites,  the  formation  of 


42  BIOLOGY  OF  BACTERIA 

peptone  from  albumins,  the  formation  of  kreatinin,  and  the  change 
of  hemoglobin  into  methemoglobin. 

Chromogenic  Bacteria. — There  are  a  number  of  both  pathogenic  and 
numerous  non-pathogenic  bacteria  which  in  their  growth  under 
certain  conditions  form  pigments  of  various  colors.  One  of  the 
conditions  necessary  to  pigment  formation  is  the  presence  of  mag- 
nesium and  sulphur  compounds  in  the  culture  medium.  Free  oxygen 
is  also  generally  required.  There  are  examples  when  free  oxygen  must 
not  be  present,  as  in  the  case  of  the  Spirillum  rubrum,  which  forms 
a  red  pigment  only  under  anaerobic  conditions.  As  a  rule,  the  pig- 
ment is  formed  better  at  lower  temperatures  than  are  present  in 
the  incubator,  and  better  in  the  dark  than  in  the  olirect  or  diffuse 
sunlight.  Some  bacteria  form  a  pigment  only  on  certain  culture 
soils,  as,  for  example,  the  glanders  bacillus  on  potatoes.  Some  chro- 
mogenic  bacteria  and  the  colors  which  they  produce  are: 

The  Staphylococcus  pyogenes  aureus,  a  golden-yellow  pigment. 

The  Staphylococcus  pyogenes  citreus,  a  lemon-yellow  pigment. 

The  anthrax  bacillus,  a  brown  pigment. 

The  glanders  bacillus  (on  potato),  a  red-brown  to  yellowish-red 
pigment. 

The  Bacillus  pyocyaneus,  a  green  pigment. 

The  Bacillus  violaceus,  a  violet  pigment. 

The  Bacillus  of  avian  tuberculosis,  a  yellowish-red  to  brown  pig- 
ment. 

The  Bacillus  prodigiosus,  a  red  pigment. 

The  Bacillus  cyanogenus  (in  milk),  a  blue  pigment. 

Some  bacteria,  particularly  those  occurring  in  sea  water,  form  a 
fluorescent  or  phosphorescent  material.  The  luminosity  or  phos- 
phorescence of  the  ocean  is  due  to  the  presence  of  these  in  enormous 
numbers. 

Fermentation  and  Enzymes. — Bacteria  and  other  low  vegetable 
microorganisms,  such  as  saccharomyces  (yeast  cells)  and  moulds, 
play  an  extensive  role  in  the  outside  world  as  the  cause  of  fermenta- 
tive processes  of  organic  compounds.  Quite  a  number  of  soluble 
ferments  or  enzymes  are  furnished  by  pathogenic  bacteria,  and  they 
lead  to  certain  manifestations  both  in  the  bodies  of  infected  animals 
and  in  artificial  culture  media. 

The  enzymes  thus  secreted  are:  diastase,  the  enzyme  which  splits 
up  starch  and  forms  maltose  (malt  sugar) ;  invertase,  which  changes 
saccharose  (a  disaccharid)  into  glucose  or  grape  sugar  or  dextrose 
(a  monosaccharid) ;  rennet,  which  precipitates  the  soluble  casein  from 
milk;  urase,  which  decomposes  urea;  lipase,  which  splits  the  fats  into 
their  component  fatty  acids  and  glycerin;  and  a  peptonizing  ferrrtent, 
which  dissolves  proteids  and  forms,  from  their  complicated  body, 
peptone  and  other  simpler  compounds.  The  action  of  the  pepton- 
izing ferment  or  enzyme  can  be  well  studied  in  artificial  cultures  pre- 
pared from  blood  serum  or  gelatin.  As  the  peptonizing  of  these 


SYMBIOSIS  43 

substances  goes  on  they  become  gradually  liquefied.  The  pepton- 
izing  and  liquefying  property  of  certain  pathogenic  bacteria  is  very 
important  from  a  diagnostic  standpoint,  and  we  therefore  divide 
pathogenic  microorganisms  into  two  groups,  peptonizing  or  liquefying 
and  non-liquefying  bacteria. 

Other  fermentative  products  which  are  formed  by  certain  enzymes 
of  pathogenic  bacteria  (colon  bacillus)  in  the  splitting  of  glucose  are 
hydrogen  and  carbon  dioxide;  also  occasionally  alcohol  and  pro- 
pionic  acid.  The  anthrax  bacillus  when  grown  in  the  presence  of 
sugar  (glucose)  forms  lactic,  formic,  acetic,  and  sometimes  succinic 
acid.  The  bacillus  of  malignant  edema,  under  anaerobic  condi- 
tions, decomposes  glucose  into  ethyl  alcohol,  formic,  butyric,  and 
lactic  acid.  The  Bacillus  lactis  aerogenes  which  occurs  in  the  intes- 
tinal tract  of  man  and  the  domestic  animals  forms  lactic  acid  as  its 
main  product  from  sugar.  Some  pathogenic  bacteria  bring  about 
putrefactive  changes  in  milk  and  other  albuminous  materials.  When 
milk  is  in  the  intestinal  tract,  however,  these  changes  do  not  take 
place  due  to  the  presence  of  growing  colon  and  lactic  aerogenes 
bacilli. 

Change  of  Reaction  in  Culture  Soil. — Bacteria  in  general  and  patho- 
genic bacteria  in  particular  require,  as  has  been  pointed  out  above, 
a  certain  delicate  reaction  of  the  medium  in  which  they  grow. 

As  a  rule,  the  medium  must  be  either  neutral  or  slightly  alkaline; 
more  rarely  slightly  acid.  Excesses  in  these  slight  degrees  of  acidity 
or  alkalinity  are  fatal.  Man)  pathogenic  bacteria  growing  in  culture 
media  change  their  reaction  considerably,  and  this  change,  partic- 
ularly in  the  presence  of  certain  substances,  may  be  so  great  that 
the  bacterium  becomes  weak  or  even  dies.  This  is  particularly  true 
when  sugar  or  glycerin  are  present.  Many  pathogenic  bacteria  form 
acids  from  these  substances  which  may  increase  the  acidity  of  the 
medium  to  such  a  degree  as  to  destroy  the  bacterial  life.  Some 
bacteria,  for  instance  the  diphtheria  bacillus,  first  increase  the  acidity 
of  the  medium  and  then  reverse  their  action,  neutralizing  the  culture 
soil,  and  finally  make  it  alkaline  to  a  certain  degree.  When  the  reac- 
tion of  a  culture  medium  is  changed,  other  changes  may  occur  depend- 
ing upon  the  change  of  reaction.  For  instance,  alkaline  milk  may 
be  made  acid  by  a  growth  of  colon  bacilli  and  the  casein  precipitated 
in  the  course  of  several  days.  This  change  is  an  important  character- 
istic of  some  bacteria  growing  in  milk,  and  is  valuable  as  one  of  the 
means  of  identification  and  diagnosis. 

Symbiosis. — It  is  found  that  some  microorganisms  when  growing 
together  in  an  artificial  culture  medium  assist  each  other  materially 
in  their  growth,  or  one  may  assist  the  other  receiving  no  benefit  itself, 
but  at  the  same  time  experiencing  no  damage  or  hindrance  in  its 
growth.  When  two  bacteria  assist  each  other  in  this  manner  we  speak 
of  a  symbiosis.  The  following  groups  of  bacilli  are  properly  classed 
under  this  head:  The  anthrax  bacillus  and  the  streptococcus;  the 


44  BIOLOGY  OF  BACTERIA 

Staphyloccus  pyogenes  aureus  and  the  influenza  bacillus;  the  diph- 
theria bacillus  and  the  streptococcus.  On  the  other  hand,  two  dif- 
ferent species  of  bacteria  when  growing  together  may  show  a  mutual 
antagonism  and  a  retarding  influence.  Such  antagonism  exists 
between  the  anthrax  bacillus  and  the  Staphylococcus  pyogenes  aureus, 
the  anthrax  and  typhoid  bacilli.  Other  bacilli  ma}  not  show  any 
influence  at  all  upon  each  other's  growth  as  the  typhoid  and  colon 
bacilli  or  as  the  cholera  spirillum  and  certain  non-pathogenic  water 
spirilla.  When  symbiotic  bacteria  invade  man  or  domestic  animals 
simultaneously  or  nearly  so,  they  are  liable  to  produce  a  very  virulent 
form  of  mixed  infection.  This  has  been  observed  in  man  in  diphtheria 
with  simultaneous  streptococcus  infection  of  the  tonsils. 

Influences  Inimical  to  Bacterial  Growth  and  Life. — We  have 
already  seen  that  bacteria  for  their  metabolism,  growth,  and  multi- 
plication require  certain  definite  conditions  of  the  nutritive  material, 
its  concentration,  moisture,  reaction,  the  prevalence  of  a  certain 
temperature,  and  the  presence  or  absence  of  oxygen.  It  has  also 
been  shown  that  any  excess  of  alkalinity  or  acidity  is  very  detri- 
mental, and  that  particularly  mineral  acids,  even  in  very  moderate 
concentration,  speedily  kill  pathogenic  bacteria.  It  has  also  been 
explained  how  their  own  accumulating  metabolic  products  bring 
about  the  same  result.  In  addition,  the  following  outside  inimical 
influences  may  be  mentioned  briefly. 

Sunlight. — Many  bacteria  can  stand  the  sunlight  quite  well;  others, 
particularly  pathogenic  bacteria,  are  rapidly  killed  by  such  exposure; 
with  them  even  diffuse  daylight,  when  acting  long  enough,  frequently 
proves  fatal. 

Electricity. — Electricity  passing  through  a  fluid  culture  medium 
forms  acids  and  alkalies  which  are  very  detrimental  to  bacteria; 
ic-rays,  as  far  as  known,  have  no  effect. 

Chemicals. — Some  chemicals,  such  as  corrosive  sublimate,  chloride 
of  lime,  permanganate  of  potash,  carbolic  acid,  creosote,  creolin, 
lysol,  formalin,  etc.,  even  in  weak  concentration,  have  a  tendency 
to  kill  bacteria  rapidly.  Chemicals  which  possess  this  property  are 
called  antiseptics  or  germicides. 

Heat. — Heat,  particularly  when  moist,  is  very  inimical  to  micro- 
organisms. As  a  rule,  most  disease-producing  bacteria  in  the  vege- 
tative form  are  killed  by  a  short  exposure  (ten  minutes)  to  a  com- 
paratively moderate  temperature  (say  55°  to  60°  C.).  This  heat,  how- 
ever, must  be  moist;  if  dry  and  the  bacteria  are  in  the  same  condition 
they  can  often  stand  much  higher  temperatures  and  longer  expos- 
ures. Spores,  however,  can  often  stand  a  long  exposure  to  moist 
heat  at  high  temperatures  (100°  C.).  When  instruments,  sutures,  and 
bandaging  material  are  exposed  to  the  heat  of  boiling  water  or  steam 
at  100°  C.,  it  is  done  with  the  object  of  killing  all  bacteria  and  their 
spores.  Such  a  procedure,  provided  it  has  been  done  successfully, 
is  called  sterilization. 


QUESTIONS  45 

Asepsis. — The  term  aseptic  refers  to  methods  and  manipulations 
which  protect  a  sterile  or  relatively  sterile  material  from  any  further 
contamination  or  admixture  with  microorganisms.  If,  for  instance, 
we  shave  the  skin  of  an  animal,  wash  and  scrub  it  thoroughly  with 
an  antiseptic  solution,  then  wash  our  hands  in  an  antiseptic  solution, 
and  finally  draw  some  blood  from  a  vein  with  a  perfectly  sterile 
hypodermic  syringe,  we  can  say  that  we  have  drawn  the  blood  in 
an  aseptic  manner.  If  everything  has  been  done  correctly,  we  have 
obtained  the  blood  without  contaminating  it  with  microorganisms. 
If  there  are  any  found  at  all  they  must  have  been  in  the  circulating 
blood  as  infecting  microorganisms. 


QUESTIONS. 

1.  At  what  range  of  temperature  can  bacteria  as  a  class  exist,  grow,  and 
multiply  ? 

2.  What  bacteria  are  most  limited  as  to  the  range  of  temperature  at  which 
they  can  multiply,  and  why? 

3.  What  is  the  optimum  temperature  of  growth  of  a  bacterium? 

4.  What  are  the  minimum  and  maximum  temperatures  of  growth? 

5.  What   is  the  optimum  temperature  for  the   (a)   mammalian,   (6)   avian 
tubercle  bacilli? 

6.  What  are  the  maximum  and  minimum  temperatures  for  these  bacilli? 

7.  What  range  of  temperature  of  growth  do  facultative  parasites  generally 
have,  and  why? 

8.  What  is  a  thermophile  bacterium  ? 

9.  Where  do  they  occur  and  where  do  they  find  the  conditions  necessary  for 
their  multiplication  ? 

10.  What  are  thermo tolerant  bacteria? 

11.  What  is  meant  by  the  thermal  death  point  of  a  bacterium? 

12.  What  do  bacteria  require  for  their  nutrition?     Can  they  grow  without 
moisture? 

13.  What  is  the  effect  of  desiccation  upon  bacteria  and  their  spores? 

14.  What  is  the  effect  of  desiccation  upon- 

(a)  The  spores  of  anthrax  and  tetanus  bacilli  ? 
(6)  The  tubercle  bacillus  ? 
(c)  The  glanders  bacillus? 

15.  What  is  meant  by  the  latent  life  of  a  bacterium  or  its  spore? 

16.  From  what  materials  can  pathogenic  bacteria  best  derive  their  food? 

17.  Name  some  pathogenic  bacteria  difficult  to  grow  on  artificial  culture  media. 

18.  Name  some  which  have  never  been  grown  successfully. 

19.  What  effect  has  too  great  a  concentration  of  solids  in  an  artificial  culture 
medium  upon  the  growth  of  bacteria? 

20.  What  effect  has  the  reaction  of  the  culture  medium  upon  the  growth  of 
bacteria  ? 

21.  What  natural  unchanged  materials  do  certain  pathogenic  bacteria  require 
before  they  will  grow? 

22.  Explain  the  terms  plasmolysis  and  plasmoptysis. 

23.  Explain  the  following  terms: 

Obligate  anaerobe.  Facultative  anaerobe. 

Obligate  ae'robe.  Facultative  ae'robe. 

24.  Name  some  pathogenic  anaerobes. 

25.  Name  some  pathogenic  aerobes. 

26.  Name  some  pathogenic  facultative  aerobes. 

27.  How  can  strict  or  obligate  anaerobes  grow  in  the  presence  of  oxygen? 

28.  What  are  some  of  the  common  metabolic  products  of  bacterial  life? 

29.  Name  a  bacterium  which  in  the  presence  of  peptone  forms  indol.    What  is 
indol? 

30.  What  is  meant  when  certain  bacteria  are  said  to  have  a  reducing  action  ? 


46  BIOLOGY  OF  BACTERIA 

31.  What  is  meant  when  certain  bacteria  are  said  to  reduce  nitrates  to  nitrites? 

32.  What  is  a  chromogenic  bacterium? 

33.  What  are  some  of  the  conditions  necessary  to  the  formation  of  bacterial 
pigments? 

34.  What  are  some  of  the  conditions  favorable  to  the  formation  of  bacterial 
pigments? 

35.  Enumerate  several  species  of  chromogenic  bacteria  and  describe   their 
pigments. 

36.  Where  are  phosphorescent  bacteria  found  ?    What  is  meant  by  this  term  ? 

37.  What  different  enzymes  are  furnished  by  various  pathogenic  bacteria? 

38.  Explain  the  action  and  name  the  fermentative  products  of  the  following 
enzymes  : 

(a)  Diastase.  (d)  Urase. 

(6)  Invertase.  (e)  Lipase. 

(c)  Rennet.  (/)   Pepsin  or  trypsin. 

39.  What  is  meant  by  the   statement  that  a  bacterium  belongs  to  the  (a) 
liquefying;  (6)  non-liquefying  type? 

40.  What  does  the  anthrax  bacillus  form  when  growing  in  a  medium  contain- 
ing sugar  ? 

41.  What   does   the   bacillus   of   malignant  edema  form  under  similar  but 
anaerobic  conditions? 

42.  What  does  the  Bacillus  lactis  aerogenes  form  from  sugar? 

43.  How  does  bacterial  growth  affect  the  reaction  of  the  culture  soil? 

44.  What  occurs  in  slightly  alkaline  milk  when  the  colon  bacillus  develops 
in  it? 

45.  What  is  meant  by  symbiotic  pathogenic  bacteria? 

46.  What  is  meant  by  bacterial  antagonism  ? 

47.  Name  some  symbiotic  and  some  antagonistic  pathogenic  bacteria. 

48.  What  generally  is  the  effect  of  direct  sunlight  upon  pathogenic  bacteria? 

49.  What  is  the  effect  of  an  electric  current  passing  through  a  fluid  culture 
medium  containing  bacteria?    Upon  what  does  the  effect  depend? 

50.  What  is  the  meaning  of  the  term  antiseptics?    Name  some  of  those  most 
commonly  employed. 

51.  What  is  the  comparative  effect  of  dry  and  moist  heat  upon  bacteria  and 
their  spores? 

52.  What  is  the  meaning  of  the  terms:  (a)  sterilization;  (6)  aseptic? 

53.  How  can  blood  be  obtained  from  an  animal  in  an  aseptic  manner? 

54.  What  is  the  meaning  of  the  term  bacterial  contamination  ? 


CHAPTER    V. 

OCCURRENCE  OF  PATHOGENIC  BACTERIA  IN  NATURE- 
ROUTES  OF  ENTRANCE  IN  INFECTION. 

MANY  pathogenic  bacteria  are  only  facultative,  and  not  strict 
parasites.  They  are  not  only  found  in  infected  persons  and  animals, 
but  also  in  the  outside  world,  where  as  saprophytes  they  find 
all  conditions  necessary  for  their  existence,  growth,  and  multipli- 
cation. There  are  a  number  of  bacteria  of  this  type  which  have  an 
extensive  distribution  in  nature  and  only  occasionally  invade  the 
organism  of  animals,  to  lead  there  a  parasitic  existence.  Among  these 
we  may  mention  the  pus-producing  (pyogenic)  staphylococci.  They 
are  truly  ubiquitous,  and  are  found  in  the  air,  soil,  water,  on  the  external 
surfaces  of  all  animals,  in  the  various  parts  of  their  gastro-intestinal 
and  respiratory  tracts,  and  on  the  outside  surfaces  of  all  objects  in 
nature,  whether  animate  or  inanimate. 

Such  organisms  as  the  tetanus  bacillus,  the  bacillus  of  malignant 
edema,  and  the  Bacillus  enteritidis  sporogenes  are  prevalent  in  the 
soil;  the  first  one  of  the  three  also  occurs  extensively  in  the  intestines 
of  the  horse  as  a  harmless  commensale.  The  ray  fungus  is  found  in 
nature  on  many  grasses,  and  the  anthrax  bacillus  on  pastures,  where 
it  multiplies  and  leads  a  saprophytic  existence. 

Certain  pathogenic  bacteria  are  only  found  in  the  outside  world  in 
the  neighborhood  of  infected  persons  and  animals  which  are  respon- 
sible for  their  dissemination.  In  this  manner  typhoid  bacilli  are 
sometimes  transferred  to  the  soil,  and  the  spirilla  of  Asiatic  cholera 
to  stagnant  waters  or  even  rivers,  notwithstanding  the  fact  that 
rapidly  flowing  rivers  generally  rid  themselves  quickly  of  pathogenic 
bacteria  through  the  effect  of  sunlight  and  by  the  aid  of  algae  which 
destroy  them.  The  anthrax  bacillus,  while  undoubtedly  found  in 
nature  independent  of  anthrax-sick  animals,  is  often  much  dissem- 
inated by  the  latter. 

There  are,  on  the  other  hand,  certain  strictly  parasitic  pathogenic 
bacteria  only  found  in  the  neighborhood  of  sick  persons  or  animals 
from  which  they  may  spread  for  a  short  distance.  Tubercle  bacilli 
and  glanders  bacilli  belong  to  this  type  and  cannot,  as  far  as  known, 
exist  in  the  outside  world.  Their  presence  in  it  is  directly  or  indi- 
rectly dependent  upon  beings  suffering  with  their  specific  diseases. 

Pathogenic  bacteria  in  the  outside  world  may  exist  in  or  on  the 
food  of  man  and  animals.  The  hay  harvested  from  anthrax-infected 
prairies  and  pastures  will  contain  this  bacillus  and  its  spores,  and 


48      OCCURRENCE  OF  PATHOGENIC  BACTERIA  IN  NATURE 

may  spread  the  disease  wherever  the  hay  is  taken.  Milk  from  tuber- 
cular cows  may  contain  the  tubercle  bacillus,  and  when  used  in  the 
raw  state  may  spread  the  disease  to  animals,  particularly  to  young 
calves  and  hogs,  and  man.  Pathogenic  bacteria  which  are  excreted 
with  the  feces,  urine,  and  other  discharges  may  contaminate  the  floors 
and  walls  of  houses,  barns,  stables,  and  also  the  bedding,  straw,  hay, 
and  manure,  and  may  exist  on  or  in  these  objects  for  a  long  time. 
In  cadavers  properly  buried  pathogenic  bacteria  do  not  remain  very 
long,  not  over  a  few  months  at  most. 

Portals  of  Entrance  for  Pathogenic  Bacteria. — Pathogenic  bacteria, 
whether  they  exist  in  nature  as  saprophytes  like  the  tetanus  or  anthrax 
bacilli,  or  are  there  temporarily  like  the  tubercle  and  glanders  bacilli, 
or  are  directly  disseminated  from  a  sick  to  a  healthy  living  being, 
must  always  enter  a  susceptible  animal  through  certain  portals  of 
entrance  in  order  to  find  the  proper  conditions  for  invasion  and 
subsequent  growth  and  multiplication.  Sometimes  a  pathogenic 
bacterium  may  gain  entrance  by  several  routes,  and  sometimes  it 
can  enter  but  by  one.  For  example,  as  far  as  known,  the  tetanus 
bacillus  never  infects  a  horse  through  the  respiratory  tract  by  inha- 
lation nor  through  the  gastro-intestinal  tract  by  ingestion.  It  must 
always,  in  order  to  produce  its  disease,  enter  through  a  wound. 
Likewise,  the  typhoid  bacillus  and  the  spirillum  of  Asiatic  cholera 
can  infect  man  only  through  the  gastro-intestinal  tract  by  ingestion. 
On  the  other  hand,  the  anthrax  bacillus  may  invade  the  tissues 
through  any  one  of  these  three  channels. 

Skin. — The  intact  skin  with  its  outer  layers  of  cornified  epithelial 
cells  forms  an  almost  perfect  barrier  against  the  invasion  of  patho- 
genic bacteria.  However,  slight  breaks  in  the  skin  are  quite  common, 
and  these  form  the  portals  of  entrance  for  a  host  of  wound  infections 
by  the  pyogenic  cocci,  the  tetanus,  anthrax,  malignant  edema, 
black-leg,  necrophorus  bacilli,  by  the  organism  of  botryomycosis. 
Sometimes  an  animal  may  even  be  infected  through  the  intact  skin, 
particularly  if  the  pathogenic  organisms  are  rubbed  in.  In  this  way 
guinea-pigs  may  be  infected  with  the  bacillus  of  bubonic  plague. 
While  open,  and  particularly  small,  deep-seated  puncture  wounds 
offer  a  very  favorable  means  of  entrance  to  many  pathogenic  bac- 
teria; it  has  been  found  that  a  wound  once  covered  with  granulation 
tissue  becomes  impenetrable.  Evidently  the  granulations  with  their 
numerous  young  connective  tissue,  vascular  endothelial  cells  and 
polyniiclear  leukocytes  form  a  barrier  which  cannot  be  broken 
through. 

Mucous  Membranes. — The  mucous  membranes  of  the  body  are 
much  less  resistant  than  the  skin.  Some  pathogenic  bacteria  can 
invade  certain  animals  even  through  an  intact  mucous  membrane. 
For  example,  if  glanders  bacilli  are  placed  on  the  conjunctiva  of  a 
guinea-pig  this  animal  contracts  glanders;  if  plague  bacilli  are  placed 
on  the  conjunctiva  of  a  rat,  the  latter  will  contract  bubonic  plague. 


PORTALS  OF  ENTRANCE  FOR  PATHOGENIC  BACTERIA   49 

Some  mucous  membranes  on  or  near  the  external  surface  of  the 
body  seem  to  possess  a  stronger  power  of  resistance  to  certain  bacteria 
than  to  others.  The  nasal  mucosa,  which  is  very  frequently  exposed 
to  the  tubercle  bacillus,  is  rarely  affected  by  it.  Tuberculosis  of  the 
nasal  mucosa  is  rare.  On  the  other  hand,  the  nasal  mucosa  of  the 
horse  cannot  offer  any  resistance  to  the  glanders  bacillus  by  which 
it  is  so  often  invaded.  Pathogenic  bacteria  frequently  find  a  portal 
of  entrance  through  the  tonsils  and  the  nasopharynx. 

Respiratory  Tract. — The  mucous  membranes  of  the  nose  and  the 
nasopharynx  are  structures  which  belong  to  the  respiratory  tract. 
This  leads  to  a  consideration  of  this  tract  as  a  portal  of  entrance. 
Infections  of  this  type  are  very  common,  and  they  occur  in  such  dis- 
eases as  tuberculosis,  glanders,  influenza,  pneumonia,  nasal  catarrh 
of  birds,  and  in  strangles  of  horses,  the  latter  caused  by  the  Strepto- 
coccus equi.  Sometimes  anthrax  and  the  pneumonic  type  of  plague 
are  also  contracted  by  inhalation. 

Gastrointestinal  Tract. — The  infection  is  brought  about  by  the 
ingestion  of  food  and  water,  in  numerous  diseases  of  man  and  the 
domestic  animals,  such  as  typhoid,  Asiatic  cholera,  anthrax,  actino- 
mycosis,  hog  erysipelas,  fowl  cholera,  tuberculosis,  bacilliary  dysen- 
tery, plague  in  rats,  various  hemorrhagic  septicemias.  The  par- 
ticular point  where  these  pathogenic  microorganisms  gain  entrance  into 
the  tissues  varies  greatly.  It  may  be  the  mucosa  of  the  mouth,  as  in 
actinomycosis  of  cattle,  or  it  may  be  the  small  intestine,  as  in  typhoid 
fever  and  Asiatic  cholera  in  man,  or  again  it  may  be  the  large  intes- 
tine, as  in  anthrax  or  tuberculosis. 

Genital  Tract. — The  genital  tract  forms  the  portal  of  entrance  for 
pathogenic  bacteria  in  sexual  intercourse.  Infectious  diseases  devel- 
oped in  this  manner  are  perhaps  more  prevalent  in  man  (gonorrhea, 
soft  chancre,  syphilis),  but  there  are  also  dieases  of  this  type  among 
the  domestic  animals.  Tuberculosis  of  the  testicles  of  the  bull  has 
given  rise  to  tuberculosis  in  the  vagina  of  the  cow,  but  this  is  exceed- 
ingly rare.  There  are,  however,  other  animal  infections  more  com- 
monly transmitted  in  sexual  intercourse.  In  cows,  infectious  abor- 
tion due  to  the  korynebacterium  abortus  infectiosi  of  Bang  is  spread 
by  the  bull  from  already  infected  to  healthy  cows.  The  latter  after 
the  abortion  harbor  and  discharge  the  bacterium  for  a  long  time, 
often  for  years,  and  abort  again  and  again,  continuing  the  spread  of 
the  disease  through  the  bull.  The  infectious  abortion  of  mares,  due 
to  a  bacillus  first  discovered  by  Ostertag,  is  likewise  spread  by  sexual 
intercourse,  and  its  entrance  is  made  thro  ugh  the  genital  tract.  Another 
bacterial  disease  which  finds  its  portal  of  entrance  in  the  genital 
organs  in  sexual  intercourse  is  the  infectious  vaginal  catarrh  of  cows 
(kolpitis  granulosa  infectiosa  bovum),  caused  by  a  specific  strepto- 
coccus discovered  by  Ostertag  and  Hecker.  Dourine  of  horses  also 
makes  its  entrance  in  a  similar  manner.  This  disease,  however,  is  not 
a  bacterial  but  a  protozoan  infection  (a  trypanosomiasis). 
4 


50       OCCURRENCE  OF  PATHOGENIC  BACTERIA  IN  NATURE 

Cryptogenetic  Origin. — Pathogenic  bacteria  may  first  invade  the 
body  through  wounds,  by  inhalation,  or  by  ingestion,  and  be  taken 
up  by  the  blood  or  lymph  current.  They  may  then  be  deposited  in  the 
lymph  glands  and  later  carried  to  other  places,  where  they  multiply 
considerably  to  produce  their  most  important  pathologic  lesions  and 
changes.  This  happens  in  glanders  where  the  important  lesions  are 
found  far  from  the  original  place  of  entrance,  and  it  occurs  also  in 
such  diseases  as  pyelonephritis  bacillosa  bovis  and  necrotic  liver 
abscesses  in  cattle  due  to  the  Bacillus  necrophorus.  Pus-producing 
microorganisms  which  sometimes  produce  very  insignificant  lesions 
at  the  portal  of  entrance  are  carried  on  and  in  another  place  produce 
profound  purulent  lesions.  This  occurs  in  bone  abscesses  (osteo- 
myelitis) and  in  abscesses  of  the  ovary.  In  fact,  in  many  cases  of 
this  kind  all  trace  of  the  small  portal  of  entrance  is  lost.  This  is 
known  as  an  infection  of  cryptogenetic  origin. 

Auto-infection. — Pathogenic  microorganisms  may  enter  through 
the  respiratory  or  gastro-intestinal  tract,  and  may  not  produce  an 
infection  at  all.  They  may  simply  remain  on  the  surface  of  the  mucous 
membranes  without  penetrating  the  tissues.  In  this  manner  they  may 
remain  latent  for  a  time,  and  then,  when  circumstances  favor  them, 
due  to  the  animal  becoming  debilitated  by  overwork,  poor  food, 
chilling,  etc.,  break  into  the  tissue,  there  multiply  and  produce  their 
specific  infectious  disease.  Sometimes  bacteria  like  the  colon  bacillus 
which  live  in  the  gastro-intestinal  tract  as  harmless  commensales 
may  acquire  pathogenic  properties,  invade  the  wall  of  the  intestine, 
the  peritoneum,  the  liver,  etc.,  and  cause  an  infectious  disease  with 
all  of  its  stages  and  features.  Such  an  infection  which  comes  from 
within,  that  is,  one  that  has  no  well-established  outside  source,  is 
called  an  auto-infection. 

Placental  Circulation. — Bacteria  may  enter  the  embryo  in  utero 
through  the  placental  circulation.  This  is  a  comparatively  rare 
occurrence  (as  far  as  bacterial  infections  are  concerned),  since  the 
maternal  and  fetal  circulations  are  separate,  and  intact  villi  will 
generally  not  permit  the  passage  of  bacteria  from  mother  to  offspring. 
However,  calf  embryos  are  frequently  infected  with  tuberculosis  from 
the  mother  through  the  placental  circulation,  and  such  infection  in 
human  syphilis  is  quite  common. 

Insects. — Biting  insects  and  other  arthropods  are  responsible  for 
a  particular  kind  of  wound  infection.  Sometimes  these  insects  which 
have  previously  fed  on  infected  material  may  spread  an  ordinary  wound 
infection.  Flies  which  have  fed  on  contaminated, material  or  cadavers 
may  also  spread  disease  by  subsequent  feeding  on  material  destined 
as  food  for  man  or  domestic  animals.  While  these  occurrences  are 
by  no  means  rare,  they  are  what  may  be  called  accidental  in  their 
nature.  There  are,  however,  some  insects  which  act  as  intermediary 
hosts  for  certain  pathogenic  microorganisms,  and,  therefore,  are  par 


QUESTIONS  51 

excellence  carriers  of  the  infection.  The  most  important  examples 
of  such  intermediary  hosts  are: 

Mosquitoes,  carrying  malaria  from  man  to  man,  or  from  birds  to 
birds. 

Tsetse-flies,  carrying  trypanosomiasis  from  animal  to  animal  or 
to  man. 

Fleas  and  lice,  carrying  bubonic  plague  from  man  to  man  or 
animal  to  animal,  or  from  animals  to  man. 

Ticks,  carrying  Texas  fever  from  cattle  to  cattle. 

Bed-bugs,  carrying  relapsing  fever  from  man  to  man. 

Summary. — It  has  now  been  shown  that  the  routes  of  entrance 
into  the  bodies  of  susceptible  animals  for  pathogenic  bacteria  are 
the  following: 

1.  Through  wounds  of  the  skin  or  mucous  membranes. 

2.  By  inhalation. 

3.  By  ingestion  with  food  or  drink. 

4.  By  intimate  contact,  particularly  by  sexual  intercourse. 

5.  Through  the  placenta!  circulation. 

Excretion  of  Pathogenic  Bacteria. — This  takes  place  in  different 
ways  according  to  the  place  of  entrance  and  the  localization  of  the 
bacteria.  An  infected  wound  may  be  open  from  the  start  and  dis- 
charge, and  disseminate  the  infecting  bacteria;  or  an  abscess  may 
have  been  formed  when  the  excretion  of  the  bacteria  will  only  take 
place  after  the  abscess  has  broken  and  established  direct  or  indirect 
communication  with  the  outside  world.  In  tubercular  infections, 
glanders,  pneumonia,  influenza,  and  strangles,  the  specific  pathogenic 
bacteria  are  discharged  through  the  natural  communicating  operi- 
ings  of  the  upper  respiratory  tract,  or  the  pathologic  products  of  these 
diseases  may  be  swallowed  and  the  bacteria  discharged  with  the 
feces.  In  anthrax  many  of  the  bacteria  are  discharged  with  the  feces, 
the  urine,  and  also  the  bloody,  foamy  fluid  flowing  from  the  nostrils. 
In  typhoid  fever,  Asiatic  cholera,  fowl  cholera,  bacillary  dysentery, 
swine  erysipelas,  the  disease  producers  are  voided  in  enormous 
numbers  with  the  feces.  The  organisms  causing  infectious  abortion 
and  infectious  vaginitis  in  cows  are  discharged  with  the  vaginal 
secretion  and  also  to  some  extent  with  the  urine;  the  latter  contains 
innumerable  specific  bacteria  in  bacillary  pyelonephritis  in  cattle. 


QUESTIONS. 

1.  Name    some    facultative    parasites  which  are  extensively  found  in  the 
outside  world  and  only  occasionally  as  pathogenic  parasites. 

2.  Name  some  obligate  parasitic  bacteria  which  are  only  found  where  they 
have  been  spread  by  infected  persons  or  animals. 

3.  Where  are  the  anthrax   bacillus   and  the  ray-fungus  found  so  that  they 
may  spread  the  disease  with  fodder? 

4.  What  are  the  portals  of  entrance  through  which  pathogenic  bacteria  may 
invade  the  body  of  a  susceptible  animal? 


52      OCCURRENCE  OF  PATHOGENIC  BACTERIA  IN  NATURE 

5.  Name  a  pathogenic   bacterium    which  generally  finds  entrance  by  one 
definite  route  only. 

6.  Name  some  pathogenic  bacteria  which  may  find  entrance  through  several 
routes. 

7.  How  does  the  intact  skin  act  toward  pathogenic  microorganisms?    How 
mucous  membranes? 

8.  Name  some  pathogenic  bacteria  which  generally  gain  entrance  into  the 
body  of  an  animal  through  wounds. 

9.  Can  animals  be  infected  through  the  intact  skin  or  mucosa,  and  if  so,  by 
what  microorganisms? 

10.  How  does  granulation  tissue  act  toward  pathogenic  bacteria? 

11.  How  does  the  nasal  mucosa  act  toward  pathogenic  microorganisms? 

12.  How  do  the  pharyngeal  tonsils  act  in  the  same  respect? 

13.  Name  some  microorganisms  invading  the  body  through  the  gastro-intes- 
tinal  tract. 

14.  At  what  point  of   the    gastro-intestinal   tract  may  they  enter  into  the 
tissues? 

15.  Name  a  disease  common  in   cattle   which  generally  makes  its  entrance 
through  the  mucosa  of  the  mouth. 

16.  Name  some  diseases    in    animals   generally  contracted  through  sexual 
intercourse. 

17.  How  can  a  bull  transfer  the  bacillus  of  infectious  abortion  to  a  cow? 

18.  Do  pathogenic    microorganisms    always  produce  their  most  important 
lesions  at  their  place  of  entrance?    If  not,  what  may  happen? 

19.  What  is  meant  by  a  cryptogenetic  infection  or  an  infection  of  crypto- 
genetic  origin? 

20.  What  is  meant  by  a  latent  infection? 

21.  What  is  meant  by  auto-infection? 

22.  Name  a  bacillus  which  ordinarily  lives  in  the  intestines  of  man  and  animals 
as  a  harmless  commensale,  but  which  may  produce  auto-infections. 

23.  How  can  bacteria  enter  the  embryo  through  the  fetal  circulation? 

24.  Name  some  insects  and  allied  animals  which  as  intermediary  hosts  may 
spread  infectious  diseases. 

25.  How  and  where  are  pathogenic  bacteria  excreted? 

26.  How  can  tubercle  bacilli  from  the  lungs  be  excreted  with  the  feces? 

27.  Name  some  diseases  of  cattle  in  which  the  pathogenic  bacteria  are  voided 
with  the  urine  or  vaginal  secretion. 


CHAPTER    VI. 

INFECTION— PHAGOCYTOSIS— OPSONINS. 
INFECTION. 

WHEN  pathogenic  bacteria  enter  an  animal  by  one  of  the  routes 
indicated,  and  multiply  in  the  cavities,  organs,  or  tissues,  and  by  so 
doing  produce  disease,  an  infection  has  taken  place,  and  the  disease 
produced  is  called  an  infectious  disease.  When  a  disease  of  this  type 
can  be  transferred  directly  from  one  animal  to  another  it  is  called 
contagious.  Tuberculosis  and  glanders,  for  instance,  are  contagious 
diseases;  but  actinomycosis,  while  infectious,  is  not  contagious, 
because  there  is  no  proof  that  it  is  ever  directly  transferred  from  one 
animal  to  another;  the  infective  and  infecting  agent,  the  actinomyces 
fungus,  being  always  taken  up  with  the  fodder.  Texas  fever  is  an 
infectious  disease,  but  it  is  not  contagious.  However,  even  purely 
infectious  non-contagious  diseases  may  be  directly  transferred  from 
the  parent  to  the  offspring  through  the  placental  circulation. 

Toxins.— The  question  arises,  How  do  microorganisms  produce 
disease?  Is  it  by  their  mere  presence?  Occasionally  bacteria  may 
so  multiply  that  they  accumulate  in  the  capillaries,  where  they  may 
lead  to  bacterial  thrombi  or  emboli;  but  this  is  very  exceptional. 
As  a  rule,  bacteria  produce  disease  by  forming  substances  highly 
poisonous  to  the  host  in  which  these  parasites  multiply.  These  sub- 
stances are  called  toxins.  When  a  venomous  snake  bites  it  is  not 
the  small  wound  which  causes  disease  and  death,  but  the  venom  which 
gets  into  the  circulation.  In  the  same  way  it  is  not  the  presence  of 
bacteria  in  the  tissues  and  juices,  but  the  toxins  which  they  manu- 
facture in  their  physiologic  processes  that,  as  a  rule,  cause  disease. 

Types  of  Toxins. — We  must  distinguish  between  two  types  of 
toxins,  the  extracellular  or  soluble  toxins,  and  the  intracellular  or 
insoluble  endotoxins. 

Some  pathogenic  bacteria,  for  instance  the  tetanus  bacillus  and  the 
diphtheria  bacillus,  in  their  growth  in  artificial  media  form  soluble 
extracellular  toxins.  These  get  into  the  fluids  in  which  the  bacilli 
grow  and  the  latter  can  be  removed  by  filtration,  so  that  a  fluid  is 
obtained  containing  the  soluble  toxins  only.  On  the  other  hand,  if 
colon,  typhoid,  or  anthrax  bacilli,  or  cholera  spirilla,  are  grown  in 
fluid  culture  media  no  toxin  will  be  found  in  the  fluid.  Finely 
divided  or  decomposed  bacteria  of  this  kind  must  be  disinte- 
grated before  it  is  possible  to  obtain  their  intracellular,  ordinarily 


54  INFECTION,  PHAGOCYTOSIS,  OPSONINS 

insoluble  toxins.  Likewise,  in  the  body  of  an  infected  animal 
such  bacteria  are  in  all  probability  subjected  to  more  or  less  dis- 
integration when  their  intracellular  toxins  are  set  free  and  exert 
their  harmful  influence.  When  pathogenic  bacteria  invade  the  body 
of  an  animal  it  may  happen  that  they  multiply  enormously  in  a  very 
short  time,  as  in  the  case  of  the  anthrax  bacillus.  Again,  they  may 
multiply  very  moderately,  but  evidently  produce  either  a  large  amount 
of  poison  or  a  very  powerful  soluble  toxin.  The  tetanus  bacillus 
is  of  this  latter  type.  Very  little  is  known  of  the  real  intrinsic  chem- 
ical nature  of  most  of  the  toxins,  and  they  cannot  be  distinguished 
by  chemical  tests.  Still,  their  dissimilarity  is  clearly  shown  by  their 
different  effects  upon  susceptible  animals  and  by  the  production  of 
different  signs  and  symptoms  which  made  it  possible  to  distinguish 
many  infectious  diseases  long  before  microorganisms  were  known. 

Virulence. — Not  only  bacteria  of  different  types,  but  also  the  same 
kind  of  bacteria  at  different  times,  in  different  countries,  and  under 
varying  conditions,  produce  diseases  which  vary  much  in  intensity 
and  in  percentage  of  mortality.  Bacteria  which  produce  a  violent 
form  of  the  disease  with  high  mortality  are  called  virulent  bacteria 
(the  condition  is  called  virulence  or  mrulency).  Often  an  epidemic 
starts  with  a  low  degree  of  virulency,  gains  more  and  more  up  to  a 
certain  point,  and  then  diminishes  again. 

The  virulence  of  bacteria  depends  more  or  less  upon  their  power 
to  multiply  rapidly  in  the  infected  animal  or  upon  their  ability 
to  produce  a  large  amount  of  very  powerful  toxins.  Virulency, 
however,  does  not  depend  upon  the  pathogenic  bacterium  itself 
alone,  but  also  upon  properties  of  different  individuals  of  the  infected 
species  of  animal,  this  variable  factor  is  known  as  individual  suscepti- 
bility. If  animals  of  the  same  species  are  all  exposed  to  one  and  the 
same  infectious  disease,  some  may  contract  it  in  a  very  violent  form, 
other  in  a  light  form,  still  others  not  at  all.  This  variability  may  depend 
upon  age,  sex,  special  breed,  color,  robustness,  weakness,  good  or 
bad  state  of  nutrition,  or  upon  factors  not  yet  recognized. 

Increase  of  Virulence. — When  pathogenic  bacteria  are  studied  ex- 
perimentally it  is  generally  found  that  their  virulency  can  be  increased 
in  the  following  manner:  A  number  of  animals  are  first  inoculated 
with  a  moderate  dose.  A  certain  percentage  get  very  sick  and  die. 
From  one  of  the  very  sick  animals  the  pathogenic  bacterium  is  ob- 
tained and  again  a  number  of  animals  are  inoculated.  This  time  a 
larger  percentage  get  very  sick  and  die.  If  this  procedure  is  con- 
tinued a  number  of  times  an  infective  bacterium  is  obtained  of  very 
great  virulency  which  will  kill  all,  or  at  least,  a  very  high  percent- 
age. Nature  does  the  same  thing  when  an  epidemic  starts  beginning 
with  a  mild  form  of  the  disease  and  developing  into  a  very  fatal, 
virulent  form.  On  the  other  hand,  at  certain  seasons,  a  fatal  epidemic 
leads  successively  to  milder  forms  of  the  disease. 


PROTECTIVE  AGENCIES  55 

Lessening  of  Virulence. — The  virulency  of  pathogenic  bacteria 
can  be  lessened  by  a  number  of  procedures,  such  as : 

1.  Inoculating  animals  which  are  only  slightly  susceptible  and 
obtaining  the  bacteria  from  them. 

2.  Growing    the    bacteria     artificially    at    temperatures    slightly 
higher  than  the  body  temperature  of  susceptible  animals;  that  is, 
generally  higher  than  the  optimum  temperature  of  such  bacteria. 

3.  Growing  bacteria  in   the  presence  of  small  amounts  of  anti- 
septics. 

4.  Growing   for    a    few  generations,   on  artificial  culture  media. 
Certain  pathogenic  bacteria  will  lose  a  good  deal  of  their  virulency 
when  cultivated  on  such  a  medium  for  some  time.     The  pneumo- 
coccus  (the  cause  of  pneumonia)  is  an  example. 

Attenuation. — Whenever  virulent  bacteria,  through  natural  or 
artificial  means  lose  some  of  their  virulency  they  are  said  to  have 
become  attenuated  and  the  process  is  called  attenuation. 

Avirulence. — When  a  virulent  bacterium  loses  all  its  virulency  and 
becomes  non-pathogenic,  it  is  said  to  have  become  avirulent.  Some 
such  avirulent  bacteria  cannot  be  made  virulent  again,  but  there  are 
others  which  can  be  made  so  by  being  introduced  successively  into 
very  susceptible  animals. 

Mixed  fcifection. — Sometimes  more  than  one  species  of  bacteria 
infects  an  animal;  this  is  known  as  a  mixed  infection.  A  symbiosis, 
previously  defined,  would  come  under  this  head,  and  such  symbiotic 
associations  may  be  very  detrimental  to  the  infected  animal  and  lead 
to  a  very  virulent  form  of  the  disease. 

The  tetanus  bacillus,  as  already  stated,  is  an  anaerobic  bacterium. 
If  a  wound  becomes  infected  with  it  and  an  aerobic  bacterium  which 
will  absorb  the  oxygen,  the  tetanus  bacillus  has  a  better  chance  to 
multiply  and  form  more  toxins,  and  a  most  virulent  form  of  the 
disease  will  result.  Streptococci  and  diphtheria  bacilli;  staphylo- 
cocci  and  colon  bacilli  often  unite  in  symbiotic  association  in  pro- 
ducing virulent  mixed  infections. 

Mortality. — All  of  the  primary  signs  and  symptoms  and  pathologic 
changes  of  infectious  diseases,  such  as  the  loss  of  appetite,  weakness, 
inability  to  work,  prostration,  elevation  of  body  temperatures,  degen- 
erative changes  in  the  organs,  disturbances  of  circulation,  etc.,  are 
due  to  the  elaborated  and  absorbed  toxins. 

Infectious  diseases  may  have  a  high  or  low  mortality,  but  there  is 
practically  no  infectious  disease  which  in  every  case  leads  to  death. 
On  the  contrary,  the  majority  of  all  such  cases  end,  fortunately,  in 
recovery. 

Protective  Agencies. — There  are  a  number  of  factors  which  protect 
the  body  against  the  invasion  and  multiplication  of  pathogenic  organ- 
isms, and  there  are  other  factors  which  limit  both  the  multiplication 
of  bacteria  and  the  amount  of  toxins  formed  in  cases  where  the 
bacteria  have  gained  entrance  to  the  body  of  the  sick  animal.  If 


56  INFECTION,  PHAGOCYTOSIS,  OPSONINS 

these  factors  had  not  been  developed  in  the  evolution  of  the  higher 
animals  the  latter  would  at  all  times  be  exposed  to  the  invasion  of 
pathogenic  bacteria,  and  toxin  production  in  an  infected  animal 
would  go  on  until  death  resulted. 

In  the  first  place,  pathogenic  bacteria  cannot,  as  a  rule,  gain  en- 
trance into  the  tissues  of  the  body  through  a  perfect  skin  or  mucous 
membrane.  Animals  constantly  inhale  and  ingest  pathogenic  bac- 
teria. However,  as  long  as  the  mucosa  of  the  respiratory  and  gastro- 
intestinal tracts  and  the  skin  are  covered  by  unbroken  layers  of 
healthy  epithelium,  pathogenic  bacteria  have  generally  no  chance  of 
penetrating  into  the  tissues.  When  breaks  do  occur  in  the  epithe- 
lium the  bacteria,  of  course,  enter  the  tissues,  and  to  counteract 
this  invasion  the  body  of  the  higher  animals  possesses  a  number  of 
protective  agencies  which  are  always  at  work  against  the  multiplica- 
tion and  the  toxic  effect  of  the  invading  microorganisms.  These  pro- 
tective agencies  are  here  considered  systematically  and  in  detail. 


PHAGOCYTOSIS. 

Metchnikoff  was  the  originator  and  is  today  the  strongest  and 
most  ardent  exponent  of  the  theory  of  phagocytosis,  which  owes  its 
firm  position  today  to  the  untiring  efforts  of  its  originator  and  his 
pupils. 

Description. — The  word  phagocyte  (derived  from  two  Greek  roots, 
means  an  eating  or  feeding  cell)  and  the  word  phagocytosis  indicate 
the  act  by  which  small  fragments  of  bacteria  are  incorporated  into 
a  phagocyte  and  therein  subjected  to  digestion  and  assimilation. 
Metchnikoff  first  observed  phagocytosis  in  low  animal  organisms  of 
the  class  tunicata,  called  actinia.  These  possess  a  common  body 
cavity,  not  yet  differentiated  into  a  thoracic  and  abdominal  cavity, 
known  as  a  celom.  If  food  or  small  particles  of  any  kind  are  intro- 
duced into  the  celom  they  float  around  unchanged  until  they  come  in 
contact  with  the  epithelial  cells  lining  the  celom  cavity.  As  soon  as 
this  occurs  the  epithelia  send  out  protoplasmic  processes,  pseudo- 
podia,  which  surround  the  small  particles,  until  finally  they  become 
completely  incorporated  in  the  cell  protoplasm.  If  suitable  they  are 
then  digested;  if  not,  they  are  expelled.  This  is  the  process  of  phago- 
cytosis as  first  observed  by  Metchnikoff. 

The  question  then  arose,  Does  anything  like  this  process  exist  in 
higher  animals?  It  was  quite  easy  to  show  this  to  be  the  case.  If 
the  blood  of  a  goose  is  defibrinated  by  whipping  it  with  a  bundle 
of  pieces  of  wood  or  wire  there  is  obtained  a  mixture  of  blood 
corpuscles  and  serum,  minus  the  fibrin  removed  by  whipping.  This 
defibrinated  blood  can  be  mixed  with  physiologic  salt  solution, 
centrifuged,  and  the  clear  supernatant  fluid  pipetted  off.  If  this  is 
done  several  times  there  is  obtained  what  is  called  in  experiments  of 


EXPERIMENTAL  DEMONSTRATION  OP  PHAGOCYTOSIS       57 

this  kind,  washed  red  blood  corpuscles.  Now,  if  a  few  cubic  centimeters 
of  these  washed  corpuscles  suspended  in  physiologic  salt  solution  are 
injected  into  the  peritoneal  cavity  of  a  guinea-pig  and  after  a  short 
time,  say  one  hour,  removed  from  the  peritoneum  together  with  an 
exudate  from  the  abdominal  cavity  which  has  been  mixed  with 
them  the  following  is  found:  The  exudate  contains  numerous  large 
mononuclear  white  blood  corpuscles  of  the  guinea-pig,  and  these 
cells  contain  many  red  bood  corpuscles  of  the  goose.  If  some  of  the 
peritoneal  exudate  is  removed  afterward  at  intervals  of  thirty  minutes, 
the  goose  corpuscles  will  be  found  more  and  more  digested  in  the 
large  mononuclear  cells  of  the  guinea-pig,  demonstrating  that  certain 
cells  of  mammalian  animals  possess  phagocytic  properties. 

In  1883  Metchnikoff  first  claimed  that  phagocytosis  protects  the 
higher  animals  against  infection  by  disease-producing  pathogenic 
bacteria.  His  opponents,  however,  showed  that  in  animals  dead  from 
anthrax,  numerous  anthrax  bacilli  were  seen  in  the  blood,  none  of 
which  were  being  taken  up  or  digested  by  phagocytic  cells.  Metch- 
nikoff then  succeeded  in  demonstrating  that  in  animals  not  sus- 
ceptible to  anthrax  such  phagocytosis  of  bacilli  takes  place  and 
that  the  lack  of  susceptibility  depends  upon  the  fact  that  the 
anthrax  bacilli  are  taken  up  and  destroyed  by  phagocytes. 

Phagocytic  Cells  (Macrophages  and  Microphages). — It  may  very  prop- 
erly be  asked,  What  kind  of  cells  of  the  higher  animals  possess  the 
power  of  phagocytosis  ?  With  a  single  exception  all  cells  which  have 
the  power  to  act  as  phagocytes  are  derived  from  the  middle  germinal 
layer,  the  mesoderm  or  mesoblast.  Among  these  cells  two  classes 
are  represented,  namely,  wandering  cells  or  leukocytes  and  fixed  con- 
nective-tissue cells.  Not  every  white  blood  corpuscle  or  leukocyte  can 
act  as  a  phagocyte.  As  a  rule,  only  some  of  the  polynuclears  and  the 
large  mononuclears  are  phagocytic.  Among  the  fixed  tissue  cells 
endowed  with  the  power  of  phagocytosis  are  the  large  mononuclear 
cells  of  the  spleen,  the  lymph  sinuses  and  the  lymphatic  endothelia  of 
the  lymph  clefts,  the  bone  corpuscles,  the  myeloplaxes  of  the  bone 
marrow,  and  other  giant  cells.  Metchnikoff  divides  all  the  phagocytic 
cells  into  macrophages  and  microphages  (large  and  small  phagocytes). 
The  former  destroy  particularly  dead  or  foreign  cells  (foreign  blood 
corpuscles)  while  the  latter  generally  destroy  bacteria.  (The  largest 
kind  of  macrophages,  the  multinuclear  giant  cells,  also  destroy  bac- 
teria.) In  general  it  can  be  stated  that  the  wandering  cells,  the 
leukocytes,  are  more  important  in  phagocytosis  than  the  fixed  cells. 

Experimental  Demonstration  of  Phagocytosis. — Phagocytosis  of  patho- 
genic bacteria  can  be  easily  demonstrated  by  numerous  experiments. 
If  a  small  amount  of  a  twenty-four-hours-old  culture  of  virulent 
anthrax  bacilli  is  injected  into  the  lymph  sac  of  a  frog,  and  if  from 
time  to  time  some  of  the  injected  fluid  is  removed  by  the  aid  of  a 
small  pointed  glass  pipette,  it  will  be  found  that  the  white  corpuscles 
of  the  frog  take  up  and  digest  more  and  more  anthrax  bacilli  until 


58 


INFECTION,  PHAGOCYTOSIS,  OPSONINS 


FIG.  24 


the  latter  have  entirely  disappeared.  It  can  also  be  shown  that  the 
anthrax  bacilli  which  the  frog's  leukocytes  take  up  are  first  alive  and 
virulent  and  remain  so  until  the  process  of  intracellular  digestion 
and  destruction  has  progressed  to  a  certain  point. 

A  similar  experiment  can  be  made  by  injecting  a  bouillon  culture 
of  anthrax  bacilli  into  a  chicken.  Here,  again,  the  leukocytes  of  the 
chicken  act  as  phagocytes  and  destroy  the  anthrax  bacilli.  However, 
if  the  temperature  of  the  chicken  is  reduced  by  immersing  its  feet  in 
cold  water,  or  by  administering  to  it  large  doses  of  chloral  or  anti- 
pyrin,  the  leukocytes  will  be  so  damaged  that  they  lose  their  phago- 

cytic  power.  They  do  not  now 
take  up  and  destroy  the  bacilli; 
these  multiply  and  the  chicken 
dies  From  anthrax.  It  can  be 
shown  that  the  lack  of  suscep- 
^*fe  A  tibility  of  pigeons  to  human 

I*  ^  9tr  k       tuberculosis  is  due  to  the  power 

/  ^jfly^    t/^rj^         M      jof  that  bird's  leukocytes  to  de- 

**  «"•  -'  ™i      stroy  tubercle  bacilli  of  human 

derivation. 

Guinea-pigs  are  ordinarily  not 
susceptible  to  an  infection  with 

x^  &ite*~7         ^e  Streptococcus  pyogenes.     If 

^1^  fa  1  cultures    are   injected   into    the 

^vjr  peritonea]  cavity  of  the  guinea- 

pig  the  cocci  are  taken  up  and 
Numerous  dipiococci  (gonococci)  inside  of    destroyed  by  phagocytes.    How- 

the  protoplasm  of  two  polynuolear  leukocytes  •      i  i 

(phagocytes).  X  1000.  (Author's  preparation.)       ever,    Very    Virulent     Cultures     OI 

streptococci  frequently  kill 

guinea-pigs,  and  it  can  be  shown  that  such  very  virulent  cocci  are 
not  destroyed  by  phagocytosis.  Rabbits  are  very  susceptible  to  the 
tetanus  toxin,  but  if  washed  tetanus  spores  free  from  toxin  are 
injected  into  the  peritoneal  cavity,  phagocytes  take  up  these  spores 
and  make  them  innocuous. 

Immunity. — All  of  these  examples  show  that  animals  without 
receiving  any  preliminary  treatment  can  protect  themselves  against 
invasion  and  multiplication  of  pathogenic  bacteria  by  phagocytosis. 
Animals  may  be  very  susceptible,  however,  to  certain  pathogenic 
bacteria  because  phagocytosis  does  not  naturally  occur,  and,  in  con- 
sequence, a  natural  immunity  does  not  exist.  Fortunately  in  these 
cases  an  artificial  immunity  can  generally  be  brought  about. 

It  is  well,  at  this  point,  to  understand  that  we  mean  by  immunity 
the  power  of  an  animal  to  resist  the  invasion  of  pathogenic  bacteria 
(see  below).  This  power  may  be  brought-  about  artificially  by  in- 
jecting into  a  susceptible  animal  less  than  a  fatal  dose  of  a  pathogenic 
bacterium  or  a  weakened,  that  is,  an  attenuated,  bacterium  or  a  weak- 
ened virus  or  vaccine. 


PREPARATION  OF  SERUM-FREE  LEUKOCYTES  59 

If,  for  instance,  such  an  attenuated  strain  of  the  anthrax  bacillus 
(anthrax  vaccine)  is  injected  into  a  susceptible  animal,  its  leukocytes, 
heretofore  non-phagocytic  with. reference  to  anthrax  bacilli,  are  en- 
dowed with  phagocytic  properties. 

Stages. — There  are  distinguished  in  phagocytosis  three  different 
stages — namely,  the  approach  of  the  phagocyte  to  the  bacterium; 
inclusion,  and  finally,  digestion,  or  destruction,  of  the  microorganisms. 

Chemotaxis. — It  is  one  of  the  most  remarkable  facts  that  phago- 
cytes possess  a  bacteriochemical  sensibility.  This  term  means  that 
phagocytes  are  attracted  toward  certain  bacteria,  as  iron  filings  are 
attracted  to  a  magnet.  It  is  easy  to  demonstrate  this  and  to  show 
how  phagocytes  can  even  wander  through  a  vessel  wall  toward  a 
place  where  certain  bacteria  have  been  injected.  This  peculiar 
attraction  which  acts  over  a  distance  is  known  as  a  positive  chemo- 
taxis  or  chemiotaxis.  It  can,  on  the  other  hand,  be  shown  that  some 
bacteria  have  a  repelling  influence  toward  phagocytes  which  causes 
them  to  flee.  This  repelling  power  is  called  negative  chemotaxis. 
When  bacteria  exert  neither  an  attracting  nor  a  repelling  influence 
upon  polynuclear  leukocytes,  we  speak  of  an  indifferent  chemotaxis. 

Aggressins. — It  is  a  very  interesting  fact  that  the  most  virulent 
strains  of  certain  bacteria,  for  instance,  very  virulent  streptococci  or 
pneumococci,  exert  a  repelling  influence  toward  the  wandering  leuko- 
cytes, and  in  this  manner  prevent  phagocytosis.  It  is  very  prob- 
able that  the  power  of  such  virulent  strains  of  pathogenic  bacteria 
depends  upon  certain  of  their  secretory  products,  and  these  have  been 
called  aggressins. 

Phagocytosis  and  Spreading  of  Disease. — In  considering  the  process 
of  phagocytosis  as  one  of  the  protecting  agencies  of  the  body  against 
the  invasion  and  multiplication  of  pathogenic  bacteria,  it  is  impor- 
tant and  necessary  to  remember  that  phagocytes  often  swallow  more 
bacteria  than  they  can  digest.  These  bacteria  remain  alive,  secrete 
their  toxins,  weaken,  and  finally  kill  the  leukocytes.  Death  of  the 
latter  may  not  occur  for  some  time,  and  meanwhile  they  may  wander 
away  after  having  engulfed  several  bacteria,  and  thus  transport  infect- 
ing bacteria  to  a  place  distant  from  their  first  point  of  entrance.  In 
other  words,  under  some  conditions  phagocytes,  instead  of  acting  for 
good,  as  is  generally  the  case,  may  do  harm  by  spreading  infection 
from  one  place  to  another. 

Preparation  of  Serum-free  Leukocytes. — Leukocytes  of  man  or 
animals  can  be  prepared  in  such  a  manner  that  they  are  free  from 
every  trace  of  blood  serum.  This  is  done  in  the  following  manner: 
About  fifteen  to  twenty  drops  of  blood  are  drawn  by  puncturing  a 
finger  or  an  ear  with  a  sterile  surgical  needle  and  the  blood  is  allowed 
to  fall  directly  into  a  glass  centrifuge  tube  which  contains  a  1^  per 
cent,  watery  solution  of  sodium  citrate.  This  solution  prevents  the 
coagulation  of  blood.  Blood  and  citrate  solution  are  mixed  by  shaking 
and  the  mixture  is  then  centrifuged  until  a  clear  upper  stratum  is 


GO  INFECTION,  PHAGOCYTOSIS,  OPSONINS 

formed.  This  is  drawn  off  with  a  pipette  and  the  centrifuge  tube  is 
now  filled  by  pouring  into  it  a  0.85  per  cent,  (physiologic)  salt  solution. 
The  tube  is  shaken  so  that  the  sediment  is  thoroughly  mixed  with 
the  salt  solution  and  again  centrifuged.  The  clear  upper  stratum  is 
once  more  drawn  off  and  the  sediment  is  once  again  shaken  with  the 
salt  solution.  After  a  final  centrifuging  the  tube  will  present  three 
layers:  a  lowest  scarlet  sediment  of  red  blood  corpuscles,  a  narrow 
grayish-red  ring  which  contains  the  leukocytes,  and  a  perfectly  clear 
top  layer  which  is  the  salt  solution.  The  latter  is  very  carefully  drawn 
off  with  a  pipette  with  rubber  bulb,  so  that  the  gray  ring  of  leukocyte 
is  not  disturbed.  After  the  salt  solution  has  been  drawn  off  the 
grayish  layer  is  carefully  drawn  into  a  small  pipette  and  at  once 
expelled  into  a  watch-glass,  which  is  covered  to  prevent  evaporation. 
The  watch-glass  now  contains  a  mixture  composed  of  salt  solution, 
suspended  leukocytes,  and  also  a  few  red  blood  corpuscles.  Their 
presence,  however,  does  not  interfere  with  the  use  of  the  suspension 
or  emulsion  of  washed  leukocytes.  When  these  are  to  be  employed 
either  in  experimental  work  or  in  order  to  ascertain  the  opsonic  index 
(see  below),  a  small  amount  of  the  suspension  is  drawn  into  a  fine 
glass  pipette.  If  a  little  air  bubble  is  now  drawn  up  and  then  an 
amount  of  bacterial  emulsion,  equal  to  the  leukocyte  emulsion,  these 
two  fluids  can  be  mixed  in  the  pipette  by  repeatedly  drawing  them  up 
and  expelling  them  on  a  clean  slide.  After  they  have  been  finally 
drawn  into  the  pipette  the  point  of  the  latter  is  sealed  in  a  flame, 
and  the  pipette  and  its  contents  can  now  be  placed  in  an  ordinary 
bacterial  incubator  or  into  a  so-called  opsonic  incubator.  The 
mixture  is  generally  left  in  the  incubator  for  one-half  hour;  then 
the  pipette  is  removed,  its  sealed  point  broken  off,  and  the  contents 
blown  onto  a  clean  slide.  The  drop  is  then  spread  with  the  margin 
of  another  clean  slide,  the  preparation  is  air  dried,  fixed  in  the  flame 
or  in  alcohol,  and  stained.  It  can  now  be  examined  with  the  oil- 
immersion  lens  of  the  microscope  like  any  other  bacterial  prepara- 
tion. If  the  test  has  been  made  as  described  above,  it  will  be  found 
on  microscopic  examination  that  very  little  if  any  phagocytosis  has 
occurred. 

Opsonins. — It  has  been  found  by  Wright  and  Douglass  that  the 
blood  serum  contains  certain  substances  which  so  prepare  bacteria 
that  they  are  subsequently  taken  up  by  phagocytes  with  avidity. 
These  substances  contained  in  the  blood  serum  have  been  called 
opsonins  (a  .  word  derived  from  a  Greek  verb  which  means  to 
prepare). 

Characteristics. — It  is  not  known  definitely  what  these  substances 
are.  Some  investigators  strongly  maintain  that  they  are  identical 
with  hemolytic  and  bacteriolytic  amboceptors  (see  below).  Other 
serum  investigators  claim  that  they  are  bodies,  sui  generis,  and  not 
identical  with  any  of  the  other  antibodies.  Be  this  as  it  may,  it  has 
been  shown  beyond  a  doubt  that  the  blood  serum  contains  certain 


EXPERIMENTAL  DEMONSTRATION  OF  OPSONINS 


61 


FIG.  25 


substances  which  prepare  bacteria  for  subsequent  phagocytosis. 
Whether  these  substances  are  originally  formed  in  the  blood  serum, 
or  whether  they  are  furnished  by  decomposing  leukocytes,  as 
Metchnikoff  and  his  followers  claim,  is  likewise  not  settled. 

Experimental  Demonstration  of  Opsonins. — The 
presence  of  these  substances  in  the  blood  serum 
can  be  shown  by  the  following  experiments.  In 
one  test  leukocytes  washed  in  physiologic  salt 
solution  and  a  freshly  prepared  bacterial  emulsion 
are  mixed  in  equal  proportions  and  placed  in 
the  incubator  for  one-half  hour.  In  a  simulta- 
neous test,  washed  leukocytes  are  mixed  with 
blood  serum  and  a  freshly  prepared  bacterial 
emulsion,  and  the  mixture  is  treated  as  above. 
After  one-half  hour,  microscopic  preparations 
are  made  from  both  mixtures.  It  will  be  found 
that  in  the  mixture  where  blood  serum  was 
lacking  phagocytosis  has  not  taken  place,  while 
in  the  other  test  where  blood  serum  was  present 
a  good  deal  of  phagocytosis  has  occurred.  This 
clearly  shows  the  influence  of  the  opsonins  of 
the  blood  serum. 


FIG.  26 


j          | 


Opsonizing  pipette  into 
which  serum,  washed  leuko- 
cytes, and  a  bacterial  emul- 
sion have  been  drawn  up. 
(Miller  in  Therapeutic 
Gazette.) 


• 


Opsonic  incubator. 


62  INFECTION,  PHAGOCYTOSIS,  OPSONINS 

Hektoen  has  shown  that  anthrax  bacilli  are  taken  up  by  the  leuko- 
cytes of  the  dog  in  the  presence  of  dog's  blood  serum.  If  the  leuko- 
cytes are  washed  several  times  with  physiologic  salt  solution,  so  that 
they  are  entirely  free  from  serum,  and  then  mixed  with  a  bacterial 
emulsion,  no  bacteria  are  taken  up  by  phagocytosis.  Hektoen  also 
demonstrated  that  the  opsonins  in  the  dog's  blood  serum  which 
bring  about  the  phagocytosis  of  anthrax  bacilli  are  destroyed  if  the 
serum  is  subjected  to  a  temperature  of  56°  to  60°  C.  for  thirty  minutes. 
The  leukocytes  themselves  are  made  unfit  for  phagocytosis  if  heated  for 
thirty  minutes  at  45°  C.,  but  these  cells  of  the  dog  contain  a  thermo- 
stabile  anthracidal  substance,  not  destroyed  by  heating  to  56°  C., 
which  can  be  extracted  with  distilled  water  after  self-digestion.  In 
working  with  anthrax  bacilli  to  determine  phagocytosis  it  is  sometimes 
difficult,  on  account  of  the  length  of  the  pseudofilaments,  to  determine 
whether  a  given  leukocyte  is  or  is  not  engaged  in  phagocytosis.  This 
difficulty  can  be  easily  overcome  by  systematic  comparisons  of  pre- 
parations from  experiments  with  or  without  dog's  blood  serum. 

Opsonic  Index. — When  the  blood  serum  of  several  healthy  persons  or 
animals  is  examined  with  reference  to  the  same  pathogenic  micro- 
organism, it  is  found  that  the  amount  of  opsonin  present  gives  a 
fairly  constant  average.  The  amount  of  the  opsonin,  of  course,  cannot 
be  estimated  directly.  Indirectly  it  is  calculated  by  the  amount  of 
phagocytosis  which  occurs  in  properly  arranged,  simultaneous,  and 
equivalent  tests.  It  can  also  be  shown  that,  as  a  rule,  in  many  chronic 
bacterial  infections  the  amount  of  opsonin  in  the  blood  serum  falls  below 
the  normal.  For  instance,  it  will  be  found  that  one  hundred  cattle 
leukocytes  with  the  mixed  blood  sera  from  five  healthy  cattle  will 
take  up  by  phagocytosis  two  hundred  tubercle  bacilli;  while  in  the 
same  experiment  with  sera  from  five  cases  of  mild  chronic  tuber- 
culosis, each  serum  being  used  separately,  the  number  of  bacilli  taken 
up  will  be  from  120  to  150.  It  is  customary  to  compare  the  number 
of  bacilli  taken  up  in  the  test  with  the  mixed  sera  from  the  healthy 
animals  or  persons  with  the  number  taken  up  in  the  test  with  a 
serum  from  an  infected  animal,  and  the  figure  obtained  by  dividing 
the  latter  by  the  former  is  called  the  opsonic  index  of  the  blood.  In 
the  example  above  the  opsonic  index  of  the  sick  animals  would  be 
-J-§~§-,  or  -|~f$,  or  -g-g-J;  that  is,  0.6  to  0.75.  As  a  rule,  the  opsonic  index 
in  chronic  bacterial  infections  is  always  low;  that  is  below  1  (one 
being  the  normal  standard). 

Vaccines. — It  has  been  shown  experimentally  that  the  opsonic 
index  for  a  pathogenic  bacterium  can  be  raised  by  injecting  into 
the  infected  person  or  animal  a  small  dose  of  a  vaccine  or  bacterine 
prepared  from  the  particular  bacterium  which  causes  the  infection. 

Vaccines  are  generally  prepared  by  obtaining  first  the  bacterium 
which  causes  the  chronic  infection  in  pure  culture,  and  then  heating 
the  bacterial  emulsion  thus  obtained  to  a  temperature  which  will  just 
kill  the  microorganisms  without  damaging  them  too  severely.  Such 


DETAILS  OF  METHOD  OF  OBTAINING  THE  OPSONIC  INDEX     63 

a  vaccine  or  bacterine  when  injected  into  a  person  or  animal  suffering 
from  the  particular  chronic  infection  and  having  a  low  opsonic  index, 
will,  during  the  first  twenty-four  hours,  generally  lower  the  index  a 
little  more.  This  is  called  the  negative  phase.  After  the  first  twenty- 
four  hours,  however,  the  index  will  rise  from  day  to  day,  until  after 
the  sixth  day  it  is  generally  considerably  above  the  normal  opsonic 
index.  This  is  called  the  positive  phase.  Then  the  index  begins  to 
drop  again.  If,  now,  on  the  sixth  or  seventh  day,  another  vaccine 
injection  is  given,  the  index  instead  of  going  below  normal  will  rise 
again  considerably  above  normal.  It  has  been  found  that,  very  fre- 
quently, simultaneously  with  the  rise  of  the  opsonic  index  the  general 
condition  of  the  sick  person  or  animal  improves  and  complete  recovery 
may  take  place.  This  vaccine,  or,  as  it  is  also  called,  bacterine  treat- 
ment, under  guidance  of  the  opsonic  index,  which  has  to  be  ascertained 
from  day  to  day,  is  an  exceedingly  laborious,  time-consuming,  and 
delicate  task,  but  it  has  been  tried  in  man  in  thousands  of  cases.  It 
has  been  found  to  bring  favorable  results  in  chronic  local  infections 
of  the  tubercle  bacillus,  the  staphylococcus,  streptococcus,  pneumo- 
coccus,  gonococcus,  colon  bacillus,  etc. 

The  treatment  is  not  applicable  in  acute  violent  infections  and 
in  very  advanced  generalized  conditions,  as  in  chronic  tuberculosis, 
acute  septicemia,  pyemia,  pneumonia,  etc. 

Details  of  Method  of  Obtaining  the  Opsonic  Index. — The  details 
of  the  pro^dure  to  obtain  the  opsonic  index  and  to  prepare  the 
bacterine  or  vaccine  in  the  case  of  a  horse  suffering  from  a  local  chronic 
staphylococcus  infection  are  as  follows:  Ascertain  by  microscopic 
examination  and  cultures  that  the  Staphylococcus  pyogenes  aureus 
(the  common  pus-producing  staphylococcus)  is  the  cause  of  the 
chronic  suppuration.  The  steps  now  necessary  to  obtain  the  opsonic 
index  of  the  horse's  blood  (for  the  Staphylococcus  pyogenes  aureus) 
are  as  follows: 

1.  Prepare  a  culture  of  the  infecting  microbes,  and  grow  in  the 
incubator  for  eighteen  to  twenty-four  hours.    Shake  well  if  in  bouillon, 
or  if  on  agar  make  a  suspension  in  salt  solution,  then  heat  in  a  water 
bath  for  one-half  hour  at  55°  C.     Shake  well  again,  centrifuge,  and 
pipette  off  some  of  the  supernatant  uniformly  cloudy  fluid  which  is 
a  homogeneous  emulsion  of  the  Staphylococcus  pyogenes  aureus. 

2.  Get  some  blood  from  a  healthy  horse,  allow  it  to  fall  into  a 
centrifuge  tube  containing  a  1.5  per  cent,  solution  of  citrate  of  sodium. 
This  fluid  will  prevent  coagulation.    Mix  well  by  shaking.     Wash  as 
described  before  in  physiologic  salt  solution;  finally,  centrifuge  well 
once  more.    Three  layers  are  formed:  the  lowest  layer  of  red  blood 
corpuscles,  a  clear  upper  fluid  layer,  and  between  them  a  thin  grayish 
red  film.     The  latter  contains  the  white  corpuscles.     Pipette  off  the 
clear  fluid.     Now,  pipette  off  the  leukocytes.      They  will  be  mixed 
with  some  red  corpuscles,  but  this  is  of  no  significance. 


64 


INFECTION,  PHAGOCYTOSIS,  OPSONINS 


3.  Get  blood  from  three  healthy  horses.     Separate  the  serum  from 
the  corpuscles  in  each  case  by  centrifuging  or  coagulation.    Mix  the 
three  sera  in  equal  proportions.    This  mixture,  in  work  with  opsonins, 
is  called  the  pool. 

4.  Get  blood  from  the  sick  horse,  and  after  coagulation,  separate 
the  serum  from  the  corpuscles. 


FIG.  27 


After  the  pipette  has  been  prepared,  its  contents  are  repeatedly  expelled  and  drawn  up  again 
in  order  to  mix  the  three  constituents  well.     (Miller.) 

FIG.  28 


After  the  pipette  has  been  in  the  incubator,  the  contents  are  blown  on  a  slide  and  the  drop  is 

drawn  out.     (Miller.) 

5.  Mix    in    equal    amounts    the   washed    white   blood    corpuscle 
emulsion   with  the  three-serum  mixture  from  healthy  horses  (the 
pool),  and  with  the   centrifuged-heated   culture  (emulsion)   of  the 
Staphylococcus  pyogenes  aureus. 

6.  Mix  in  another  small  pipette  equal  amounts  of  the  white  blood 
corpuscle  emulsion  of  the  serum  of  the  sick  horse  and  of  the  cen- 
trifuged  heated   bouillon  culture  of   the   Staphylococcus   pyogenes 
aureus. 


DETAILS  OF  METHOD  OF  OBTAINING  THE  OPSONIC  INDEX     65 

7.  Now  place  pipettes  No.  1  (three  healthy  horses'  serum  mixture — 
the  pool)  and  No.  2  (serum  from  sick  horse)  in  the  incubator  for  one- 
half  hour. 

8.  Make  a  smear  preparation  from  pipette  No.  1  and  also  from 
No.  2.     Dry,  fix,  and  stain. 

9.  Count  200  leukocytes   in  specimen   No.    1,   and  count  how 
many  cocci  have  been  taken  up  by  phagocytosis  in  the  200  leukocytes. 

10.  Do  the  same  with  No.  2. 

FIG.  29 


The  smear,  ready  to  be  air-dried,  fixed  and  stained.     (Miller. 

The  result,  for  example,  may  be  as  follows:  200  leukocytes  in  No.  1 
have  taken  up  300  cocci  (this  is  the  mixture  of  the  sera  of  three 
normal  horses);  200  leukocytes  in  No.  2  (sick  horse's  blood  serum) 
have  taken  up  150  cocci;  therefore,  taking  the  normal  opsonic 
index  as  1  (one),  the  low  opsonic  index  of  the  serum  of  the  sick 
horse  is  0.5. 

When  it  is  found  that  the  opsonic  index  is  low,  vaccine  treatment 
(in  our  case  the  Staphylococcus  pyogenes  aureus  vaccine)  is  indi- 
cated. 

FIG.  30 


Wright's  blood  capsule  filled  with  blood  and  ready  to  be   centrifugalized   for  separating  the 
serum  from  the  clot.    (Miller.) 

In  the  above  steps,  to  determine  the  opsonic  index,  small  U-shaped 
tubes,  or,  according  to  Wright,  peculiar  glass  capsules  are  used  for 
collecting  the  blood  sera.  The  mixing  of  the  emulsion  of  leukocytes 
of  the  sera  and  the  bacterial  emulsion  is  done  in  small  pipettes.  The 
fluids  are  drawn  up  in  equal  amounts,  first  one,  then  a  little  air  bubble, 
then  the  second,  and  so  forth.  The  three  different  fluids  (serum, 
5 


66  INFECTION,  PHAGOCYTOSIS,  OPSONINS 

leukocytes,  bacterial  emulsion)  are  mixed  by  being  several  times 
drawn  up  and  again  expelled  from  the  pipette  into  a  watch  crystal 
or  glass  slide.  The  mixtures  in  the  little  pipettes,  after  these  have 
been  sealed  in  the  frame,  are  finally  placed  in  the  incubator  or  a 
special  opsonic  oven.  After  half  an  hour  the  mixtures  are  blown  on 
a  slide,  spread  out,  air  dried,  stained,  and  then  the  bacteria  taken 
in  by  200  leukocytes  are  counted. 

FIG.  31 


Opsonic   pipette  after  mixture   of   the   three   constituents;    the  tip  has  been  fused  and  the 
pipette  is  ready  for  the  opsonic  incubator.     (Miller.) 

Preparation  of  Vaccines. — Considerable  care  has  to  be  exercised  in  the 
preparation  of  the  bacterial  vaccines  or  bacterines  used  for  therapeutic 
injections.  Unless  an  autovaccine,  that  is,  a  vaccine,  from  the  bacteria 
infecting  the  patient  it  wanted  the  vaccines  are  generally  procured  from 
one  of  the  pharmaceutical  houses  which  prepare  them  in  their  biologi- 
cal laboratories.  The  autovaccine  must  be  prepared  directly  from  the 
infecting  organism.  As  a  first  step,  a  pure  culture  must  be  obtained, 
which  may  be  raised  in  bouillon  or  on  slanted  agar;  the  latter  is  prob- 
ably the  better.  The  growth  is  then  removed  with  a  platinum  loop 
or  spatula,  mixed  with  physiologic  salt  solution,  shaken  long  and 
thoroughly,  and  then  heated  to  kill  the  bacteria.  The  remaining 
coarser  particles  are  removed  by  centrifuging  and  the  uniform  emul- 
sion is  standardized  by  estimating  the  number  of  bacteria  present  per 
cubic  centimeter.  This  is  done  by  collecting  and  mixing  one  part  of 
the  vaccine,  three  of  physiologic  salt  solution,  and  one  of  normal 
human  blood  in  a  blood  pipette.  After  these  ingredients  have  been 
thoroughly  mixed,  drops  are  blown  on  a  clean  slide,  spread,  air  dried, 
fixed,  and  stained.  Then  200  red  blood  corpuscles  are  counted  and 
the  number  of  bacteria  in  the  same  number  of  fields  is  ascertained. 
Since  it  is  known  that  each  cubic  millimeter  of  normal  human  blood 
contains  five  million  erythrocytes,  the  number  of  bacteria  present 
in  the  same  amount  of  fluid  can  easily  be  calculated  from  the  number 
counted  in  the  same  spaces  which  contained  the  200  erythrocytes. 
After  the  calculation  has  been  made  the  vaccine  can  be  so  diluted 
that  it  contains  from  50,000,000  to  300,000,000  bacteria  per  cubic 
centimeter.  These  are  the  average  doses  used  in  the  vaccine  treat- 
ment in  man.  A  small  amount  of  lysol  or  some  other  antiseptic 
should  be  added  to  the  vaccine,  unless  it  is  used  at  once.  In  count- 
ing the  blood  corpuscles  and  bacteria,  it  is  advantageous  for  accurate 
work  to  have  the  microscopic  field  of  vision  limited  by  placing  horse- 
hairs in  the  form  of  a  rectangle  in  the  eyepiece  or  by  the  use  of  3 
square  diaphragm. 


PREPARATION  OF  VACCINES  67 

The  task  of  preparing  vaccines  and  ascertaining  the  daily  opsonic 
index  is  so  delicate  and  time-consuming  that  it  is  out  of  the  question 
for  the  practitioner  to  do  the  work  himself.  If  he  wants  to  treat 
cases  of  chronic  bacterial  infections  by  the  vaccine  or  bacterine 
method,  he  must  first  ascertain  what  particular  microorganism 
does  the  harm,  and  then  get  a  vaccine  prepared  from  the  identical 
species  and  use  it  by  hypodermic  injection  every  sixth  or  seventh 
day.  It  is  best  to  start  with  a  minimum  dose  and  watch  its  effect 
upon  the  animal  treated.  It  should  be  remembered  that  general 
improvement  frequently,  but  not  always,  is  coincident  with  a  rise 
in  the  opsonic  index. 

FIG.  32 


Showing  a  microscopic  field  of  the  vaccine-dilute  blood  mixture,  prepared  in  order  to 
standardize  the  vaccine.    (Miller.) 

Archibald,  in  a  paper  read  before  the  1909  Chicago  Meeting  of 
the  American  Veterinary  Association,  and  printed  in  the  Trans- 
actions of  the  Association,  stated  that  he  had  very  good  success  with 
autogenic  vaccines  prepared  by  himself  in  the  treatment  of  quittors, 
fistulse,  and  other  infective  troubles. 

Opsonins  are  specific  bodies,  which  means  that  the  injection  of  a 
staphylococcus  vaccine  will  raise  a  low  opsonic  index  for  staphylococci 
but  not  a  low  index  for  tubercle  bacilli  or  any  other  bacterium. 


68  INFECTION,  PHAGOCYTOSIS,  OPSONINS 


QUESTIONS. 

1.  What  is  an  infectious  disease? 

2.  What  is  a  contagious  disease  ? 

3.  Name  some  infectious  diseases,  also  some  contagious  diseases. 

4.  How  do  microorganisms  produce  disease? 

5.  What  is  a  toxin,  a  soluble  toxin,  and  an  intracellular  or  endotoxin? 

6.  Name  some  pathogenic  bacteria  which   form    soluble  toxins.     How  can 
they  be  obtained  free  from  bacteria  ? 

7.  Name  some  bacteria  which  form  only  insoluble   endotoxins.     How   are 
the  latter  set  free  ? 

8.  Name  a  pathogenic  bacterium  which  multiplies  very  freely  in  the  body 
and  one  which  multiplies  sparingly,  but  produces  a  powerful  soluble  toxin. 

9.  What  is  meant  by  a  virulent  bacterium  and  by  virulency? 

10.  What  is  meant  by  individual  susceptibility? 

1 1 .  What  is  meant  by  attenuation  ? 

12.  What  methods  can  be  employed  to  attenuate  virulent  bacteria? 

13.  What  is  meant  by  an  a  virulent  bacterium? 

14.  What  is  a  mixed  infection  ? 

15.  What  is  symbiosis? 

16.  Why  does  not  an  infectious  disease  attack  every  individual?    Why  does  it 
not  always  kill? 

17.  What  is  a  phagocyte?    What  is  phagocytosis? 

18.  Who  first  studied  phagocytosis,  and  in  what  animals? 

19.  What  happens  if  we  inject  washed  goose-blood  corpuscles  into  the  peri- 
toneal cavity  of  a  guinea-pig? 

20.  What  is  the  procedure  of  washing  mammalian  or  avian  blood  corpuscles? 

21.  What  kind  of  cells  of  higher  animals  possess  the  power  of  phagocytosis? 

22.  What  are  macrophages?    What  are  microphages  ?     How  do  they  differ  as 
to  their  phagocytic  properties? 

23.  How  can  the  phagocytosis  of  anthrax  bacilli  be  demonstrated? 

24.  How  do  very  virulent  streptococci  act  with  reference  to  phagocytosis? 

25.  What  is  meant  by  immunity? 

26.  What  is  an  attenuated  virus  or  vaccine? 

27.  What  are  the  three  stages  of  phagocytosis? 

28.  What  is  positive,  negative,  and  indifferent  chemotaxis? 

29.  What  is  an  opsonin? 

30.  What  is  the  effect  of  washed  leukocytes  upon  a  bacterial  emulsion?   What 
is  their  effect  in  the  presence  of  blood  serum  ?    Why  is  this  effect  produced  ? 

31.  Describe  comparative  experiments  to  show  the  effect  of  the  absence  or 
presence  of  serum  upon  phagocytosis. 

32.  Describe  such  a  set  of  experiments  with  dog's  leukocytes  and  anthrax 
bacilli. 

33.  Are  opsonins  present  in  normal  healthy  animals  in  very  variable  or  in 
rather  constant  amounts? 

34.  How  can  the  amount  of  opsonin  present  be  estimated? 

35.  What  about  the  amount  of  opsonin  in  chronic  bacterial  infections? 

36.  What  is  meant  by  the  opsonic  index?     How  is  it  estimated? 

37.  If  in  the  mixture  of  healthy  sera  in  one  experiment  2001  eukocytes  have 
taken  up  250  bacteria,  and  in  the  experiment  with  the  serum  of  the  sick  person 
or  animal  only  100  bacteria  have  been  taken  up  by  phagocytosis,  what  is  the 
opsonic  index  of  the  latter  serum? 

38.  What  is  the  effect  of  vaccine  or  bacterine  injection  upon   the  opsonic 
index  ? 

39.  What  is  the  negative  phase  after  vaccine  injection?     What  is  the  positive 
phase  ?     How  long  does  the  latter  generally  last  ?     What  occurs  then  ? 

40.  Describe  in  detail  how  to  obtain  the  opsonic  index  in  case  of  a  chronic 
staphylococcus  infection  in  a  horse 


CHAPTEE    VII. 

ANTIBODIES— IMMUNITY— EHRLICH'S  SIDE-CHAIN  THEORY— 
THE  WASSERMANN  SERUM  TEST. 

ANTIBODIES. 

Toxins  and  Antitoxins. — It  has  already  been  stated  that  some 
pathogenic  bacteria  secrete  very  powerful  soluble  toxins  which  enter 
the  general  circulation.  Whenever  such  toxins  circulate  in  the  blood 
there  is  a  tendency  to  the  formation  of  bodies  which  neutralize  them, 
and  bring  about  a  cure,  provided  that  the  toxins  are  not  overabun- 
dant and  have  not  already  done  irreparable  damage.  When  tetanus 
toxins  are  in  the  system  of  a  horse  they  are  usually  generated  so 
quickly  and  abundantly  that  before  they  can  be  neutralized  by  natural 
or  artificial  means  irreparable  damage  has  been  done  to  some  part  of 
the  body,  generally  the  central  nervous  system.  The  bodies  which 
neutralize  the  soluble  toxins  are  called  antitoxins.  Their  effect  can 
be  best  understood  by  comparison  with  the  well-known  chemical 
reaction  between  acids  and  alkalies.  There  is  a  disease  common  in 
man,  and  sometimes  found  in  the  domestic  animals,  which  is  char- 
acterized clinically  by  the  secretion  of  sugar  in  the  urine  and  by 
the  inability  of  the  body  to  properly  split  up  and  utilize  this  sugar. 
Consequently,  sugar  finds  its  way  into  the  blood,  and  from  this  carbo- 
hydrate a  large  amount  of  organic  acid  is  formed.  No  being  could 
exist  with  a  large  amount  of  free  acid  in  the  blood,  so  the  system  at 
once  corrects  the  defect  by  furnishing  to  the  blood  a  large  amount 
of  ammonia  to  neutralize  the  organic  acids.  Somewhat  similarly  the 
antitoxins  neutralize  the  toxins.  Just  as  hydrochloric  acid  can  be 
neutralized  in  the  test-tube  with  ammonia,  so  can  a  soluble  toxin 
be  neutralized  with  its  antitoxin.  The  principle  is  the  same,  although 
the  process  is  a  much  more  complicated  one  than  the  neutralization 
of  an  acid  by  an  alkali.  The  toxin-antitoxin  mixture  can  be  injected 
into  a  susceptible  animal  without  producing  any  ill  effects.  Thus 
the  formation  of  antitoxins  is  another  means  by  which  the  body 
protects  itself  against  pathogenic  bacteria;  that  is,  against  one  of 
their  most  important  products,  the  toxins. 

Agglutinins,  Lysins,  Precipitins. — If  cholera  spirilla  are  injected 
into  the  peritoneal  cavity  of  an  animal  which  is  not  susceptible  to 
them,  and  from  time  to  time  removed  by  the  aid  of  small  capillary 
pipettes,  it  will  be  noticed  that  the  spirilla  soon  lose  their  motility, 
become  glued  to  each  other,  forming  small  lumps,  indistinct  in  out- 


70       ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

line,  and  are  finally  completely  dissolved.     These  occurrences  are 
called: 

Immobilization  (loss  of  motility). 

Agglutination  (becoming  glued  together). 

Lysis  (solution). 

It  can  be  shown  that  all  of  these  processes  favorable  to  the  infected 
animal,  but  detrimental  to  the  pathogenic  infecting  bacteria,  are 
brought  about  by  definite  bodies  contained  in  the  blood  serum  and 
juices  of  the  system.  These  substances  are  known  as  agglutinins  and 
lysins.  If  human  blood  serum  is  injected  repeatedly  into  the  body  of 
a  rabbit  at  intervals  of  ten  to  fourteen  days,  and  some  time  after  the 
injection  a  little  of  the  rabbit's  blood  serum  is  obtained,  it  will  be 
found  that  a  very  small  amount  of  the  rabbit's  blood  serum  added 
to  a  very  dilute  solution  of  human  blood  will  cause  a  clouding  or  pre- 
cipitation of  exceedingly  fine  flocculi.  Something  has  evidently  been 
formed  in  the  rabbit's  blood  serum  which  precipitates  something  from 
the  human  serum.  The  body  or  bodies  so  formed  in  the  blood  serum 
of  one  animal  treated  with  the  serum  of  another  animal  are  called 
precipitins. 

The  specific  test  with  the  blood  of  a  rabbit  sensitized  against  human 
blood  serum  is  a  very  important  one  from  a  medicolegal  or  forensic 
standpoint,  because  it  makes  possible  to  identify  human  blood  stains 
in  criminal  cases  and  to  differentiate  them  from  the  blood  of  any 
other  animal.  This  test  is  much  more  delicate  than  measuring  the 
erythrocytes  in  order  to  distinguish  between  human  and  other  red 
blood  corpuscles. 

The  three  bodies,  agglutinins,  lysins,  and  precipitins,  like  opsonins 
and  antitoxins,  belong  to  the  protective  substances  of  the  animal 
body  against  pathogenic  bacteria.  A  common  name  for  all  of  these 
bodies  inimical  to  pathogenic  bacteria  is  antibodies.  Many  anti- 
bodies are  normally  present  in  higher  animals,  but  often  only  to  a 
small  extent  and  sometimes  not  at  all.  However,  the  system  can  be 
stimulated  to  manufacture  antibodies  when  not  present,  or  to  in- 
crease enormously  those  present  to  a  small  extent,  by  the  injection 
of  either  live  or  dead  pathogenic  bacteria.  Such  injections  must 
be  practised  with  certain  precautions  and  with  the  observation  of 
certain  rules,  otherwise  instead  of  strengthening  the  animal  against 
bacterial  invasions,  it  will  be  killed.  The  effect  of  injecting  live  or 
dead  bacteria  into  an  animal  is  very  different  from  injecting  certain 
chemical  poisons.  An  animal  can  be  accustomed  to  stand  succes- 
sively increasing  doses  of  morphine,  strychnine,  arsenic,  etc.,  but  in 
spite  of  such  treatment,  no  antibodies  to  morphine,  strychnine,  or 
arsenic  are  formed.  The  antibodies  formed  when  pathogenic  bacteria 
are  injected  are  specific,  i.  e.,  when,  for  instance,  tetanus  bacilli  are 
injected  antibodies  are  formed  against  tetanus  bacilli  and  their 
toxins,  but  they  have  no  effect  on  diphtheria,  glanders,  or  anthrax 
bacilli.  If  it  is  desired  to  form  antibodies  against  a  certain  bacillus, 


VACCINES  AND  ANTITOXIC  SERA  71 

this  same  bacillus  must  always  be  injected.  Antibodies  are  not  only 
formed  against  bacteria,  but  against  other  organized  material.  If, 
for  instance,  human  blood  serum  is  injected  into  a  rabbit,  there  will 
be  formed  antibodies  against  human  serum  in  the  rabbit's  blood 
serum,  and  if  sheep's  blood  corpuscles  are  injected  into  a  rabbit 
there  will  be  formed  in  the  latter 's  blood  serum  antibodies  against 
sheep's  blood  corpuscles,  etc. 

Antigens. — Any  body,  be  it  a  bacterium,  an  animal,  or  a  vegetable 
cell  or  other  organic  product  of  any  kind,  that  is  injected  into  an 
animal  by  the  paraenteral1  route  and  causes  the  formation  of  anti- 
bodies is  called  an  antigen  (meaning  to  produce  antibodies). 

Vaccines  and  Antitoxic  Sera. — The  term  vaccine  (also  vaccination) 
is  derived  from  the  Latin  word  vacca  (cow).  It  was  first  used  for  the 
procedure  of  inoculating  superficially  the  arm  of  a  person  with  the 
contents  of  a  cowpox  vesicle  or  the  contents  of  the  pustule  from 
another  person.  This  method,  as  is  well  known,  was  and  is  used  to 
protect  persons  so  vaccinated  against  smallpox.  Today  the  term 
vaccine  is  used  in  a  general  sense  for  any  infecting  living  virus  (bac- 
terium, protozoan,  or  invisible  infectious  microorganisms,  or  their 
toxins),  either  in  full  strength  (virulency),  in  a  very  small  does,  or 
in  an  attenuated  form  in  a  larger  dose,  for  the  purposes  of  producing 
antibodies;  that  is,  for  the  purpose  of  preventing  or  curing  disease. 
Some  pathogenic  microorganisms  may  even  be  killed  by  high 
degrees  of  heat  and  still  be  effective  as  vaccines  in  the  production 
of  antibodies. 

Preparation. — Reference  has  been  made  to  methods  by  which 
pathogenic  bacteria  can  be  attenuated  in  order  to  be  used  in  the 
preparation  of  vaccines.  These  various  methods  are: 

1.  Temperatures  moderately  higher  than  the  optimum — anthrax 
vaccine. 

2.  Temperatures  which  will  kill  the  bacteria — tuberculin,  black-leg 
vaccine,  staphylococcus  vaccine,  streptococcus  vaccine,  pneumococcus 
vaccine,  colon  bacillus  vaccine,  plague  bacillus  vaccine. 

3.  The  addition  of  antiseptics  in  small  amounts — carbolic  acid, 
for  anthrax  vaccine;  iodine,  for  tetanus  vaccine. 

4.  Drying — rabies  virus. 

5.  Digesting  the  bacterium — cholera  spirillum  vaccine. 

6.  Prolonged  cultivation  in  artificial  media — fowl  cholera  vaccine. 
Vaccines  are,  as  seen  above,  generally  prepared  artificially  outside 

of  the  animal  body.  Antitoxins,  however,  can  only  be  prepared  in 
the  body  of  an  animal.  The  principle  of  the  procedure  is  generally 
as  follows:  Inject  into  an  animal,  say  a  horse,  successively  increas- 
ing doses  of,  for  example,  a  diphtheria  toxin,  and  finally  larger 
amounts  of  cultures  of  virulent  diphtheria  bacilli  themselves.  These 

1  A  paraenteral  method  is  one  of  incorporation  of  a  substance  into  an  animal  subcutaneously, 
intraperitoneally,  subdurally,  intravenously,  or  by  any  other  route  than  thegastro-intestinal 
tract. 


72     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

injections  have  to  be  made  at  suitable  intervals  and  a  new  one  must 
not  be  made  before  the  effects  of  the  previous  one  (prostration,  fever, 
etc.)  have  entirely  disappeared.  By  this  method  there  is  manufac- 
tured in  the  body  of  the  animal,  i.  e.,  in  its  blood  serum,  an  enormous 
amount  of  antitoxin.  After  the  proper  prolonged  treatment  the  blood 
can  be  drawn  off  and  the  serum  separated  from  the  clot  (everything 
thing  should  be  done,  of  course,  under  aseptic  precautions). 

Antitoxic  Units. — The  serum  so  obtained  is  called  an  antitoxin, 
an  antitoxic,  or  an  immune  serum.  It  is  necessary  to  ascertain  the 
value  of  an  antitoxic  serum.  This  is  done  in  animal  experiments  in 
which  both  toxins  and  the  antitoxic  serum  to  be  tested  are  used.  The 
animals  employed  in  the  experiments  are  generally  guinea-pigs. 
The  unit  measure  of  an  antitoxin  generally  employed  is  that  amount 
which  will  protect  a  guinea-pig  of  about  one-half  pound  weight 
against  ten  times  the  ordinary  fatal  dose.  For  instance,  it  is  found 
that  0.01  c.c.  of  a  strong  diphtheria  toxin  will  kill  a  medium-sized 
guinea-pig.  Then  take  0.1  c.c.  of  the  strong  toxin  and  ascertain  the 
amount  of  antitoxin  necessary  to  neutralize  it.  Suppose  it  is  0.0025 
c.c.  Then  this  0.0025  c.c.  of  the  antitoxic  serum  is  said  to  contain 
one  immunizing  unit,  or  1  c.c.  of  this  antitoxin  contains  250  immuniz- 
ing units.  If  in  a  case  of  diphtheria  in  a  cat  it  is  known  from 
experience  that  it  will  take  500  immunizing  units  to  cure  it,  it  will 
be  necessary  to  inject  2  c.c.  of  the  antitoxic  serum  or  use  a  serum  four 
times  as  strong  and  give  0.5  c.c.  to  get  the  same  effect.  In  order  to 
protect  a  horse  prophylactically  against  tetanus  it  is  necessary  to 
give  10  c.c.  to  20  c.c.  of  a  strong  tetanus  antitoxin,  which  dose 
contains  several  thousand  immunizing  units. 

IMMUNITY. 

Definition. — Immunity  may  be  defined  as  the  ability  of  a  higher 
animal  organism  to  resist  invasion  by,  and  multiplication  of,  patho- 
genic microorganisms  and  to  neutralize  their  poisonous  products. 
The  agencies  to  which  the  protection  is  due  have  already  been  named 
and  explained.  They  are  the  phagocytic  cells,  the  opsonins,  anti- 
toxins, agglutinins,  lysins,  precipitins,  and  a  number  of  others. 

Congenital  Immunity. — Certain  animals  may  possess  a  congenital 
natural  immunity.  For  instance,  many  warm-blooded  mammals, 
such  as  cattle,  sheep,  mice,  rabbits,  guinea-pigs,  are  susceptible  to 
anthrax  infection,  while  adult  dogs  and  rats  possess  quite  a  strong, 
though  not  absolute,  natural  immunity  against  this  infection.  The 
horse  and  man  are  susceptible  to  glanders;  cattle  are  immune. 
Man  is  susceptible  to  typhoid  bacillus  and  cholera  spirillum  infec- 
tions, while  all  our  domestic  animals  are  immune,  as  far  as  natural 
infection  is  concerned. 

Acquired  Immunity. — Persons  who  have  one  attack  of  the  following 
diseases  are  generally  immune  against  a  second  attack,  viz.,  measles, 


BENZENE  RING  73 

scarlatina,  typhoid  fever,  smallpox.  The  same  is  true  of  animals 
after  once  having  had  hoof-and-mouth  disease,  rinderpest,  and  dis- 
temper (in  dogs).  This  is  called  a  natural  acquired  immunity. 

Immunity  may  also  be  acquired  by  artificial  means.  When  an 
animal  is  inoculated  with  an  attenuated  virus  or  vaccine  the  aim  is 
to  produce  a  comparatively  mild  attack  of  the  disease.  This  mild 
attack  protects  the  animal  for  some  time  against  another  attack  of 
the  same  infection,  be  it  severe  or  otherwise.  This  procedure,  known 
as  (artificial)  active  immunity,  has  made  the  animal  immune. 
Example,  anthrax  vaccination  with  the  attenuated  virus. 

Passive  Immunity. — Besides  vaccination,  immunity  may  also  be 
conferred  by  injecting  into  an  animal  the  antitoxic  or  immune  serum 
of  another  animal.  This  type  is  called  passive  immunity,  and  is 
largely  used  in  preventing  tetanus  or  diphtheria  by  the  subcutaneous 
injection  of  tetanus  and  diphtheria  antitoxins. 

Simultaneous  Method. — In  some  cases  an  active  immunity  is  pro- 
duced by  the  so-called  simultaneous  method,  that  is,  by  an  injection 
of  a  virus  or  vaccine  and  an  antitoxin  at  the  same  time.  An  active 
immunity  generally  lasts  much  longer  and  protects  much  better  than 
a  passive  immunity,  but  in  some  cases  it  is  quite  dangerous  to  inject 
even  a  very  small  but  still  effective  dose  of  a  virus.  Therefore,  in 
order  to  lessen  the  danger  of  severe  sickness  and  death  the  antitoxin 
is  injected  at  the  same  time.  The  simultaneous  method  is  employed 
in  the  following  cases:  In  inoculating  horses  for  tetanus  with  both 
tetanus  toxin  and  antitoxin;  in  immunizing  cattle  against  rinder- 
pest by  injecting  simultaneously  blood  from  a  virulent  case  and  serum 
from  an  animal  that  has  recovered  from  the  disease;  by  immunizing 
cattle  against  anthrax  by  injecting  at  the  same  time  an  attenuated 
bacillus  and  the  immune  serum  from  a  mule.  This  method  is  also 
used  in  immunizing  swine  against  hog  cholera. 

Ehrlich's  Side-chain  Theory  of  Immunity. — It  has  previously 
been  stated  that  toxin  and  antitoxin  apparently  unite  inside  and 
outside  of  the  body,  neutralizing  each  other  somewhat  like  ordi- 
nary chemicals,  such  as  acids  and  alkalies.  It  can  be  shown  that 
the  experimental  union  of  toxin  and  antitoxin  always  takes  place 
in  definite  amounts,  and  that  it  occurs  more  quickly  in  concen- 
trated solutions  and  at  higher  temperatures  (35°  to  40°  C.).  These 
facts  led  Ehrlich  to  suspect  that  the  chemically  highly  complex 
toxins  and  antitoxins  possess  certain  molecular  groups  having  toward 
each  other  a  high  degree  of  chemical  affinity,  which  causes  them  to 
unite  somewhat  as  acids  and  alkalies  do  whenever  there  is  a  chance 
for  a  union.  From  this  fundamental  idea  Ehrlich  developed  his 
celebrated  side-chain  theory  of  immunity.  In  order  to  understand 
this  it  is  well  first  to  explain  what  is  meant  by  a  side-chain  in  organic 
chemistry. 

Benzene  Ring. — According  to  the  hypothesis  of  Kekule,  the  organic 
compounds  of  the  aromatic  group  of  which  benzene  or  benzole  is  a 


74     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM   TEST 

representative,  have  their  carbon  atoms  arranged  in  a  ring.  Carbon 
is  a  tetravalent  atom,  that  is,  each  atom  has  four  chemical  affinities 
which  can  be  satisfied  by  other  atoms  in  forming  various  chemical 
compounds.  The  formula  of  benzene,  which  is  a  stable  chemical 
compound,  in  which  all  affinities  are  satisfied,  is  C6H6.  Its  chemical 
structure,  according  to  Kekule's  hypothesis,  is  explained  by  assuming 
that  the  tetravalent  carbon  atoms  form  a  ring  in  which  each  of  the 
carbon  atoms  is  united  with  one  neighbor  by  two  affinities  and  with 
the  other  neighbor  by  one  affinity.  This  leaves  for  each  carbon 
atom  one  affinity  free  to  which  the  hydrogen  atom  is  united.  The 
picture  of  the  hypothetical  benzene  ring  is  shown  in  Formula  A. 

H  H 


H— C        C— H  H— C        C— NH2 

H— C        C— H  H— C        C— H 


Formula  A.      Benzene.  Formula  B.      Anilin  oil. 

Each  of  the  hydrogen  atoms  may  be  replaced  by  a  more  complicated 
molecular  group,  so  that  we  may  have,  for  instance,  a  body  of  the 
formula  C6H5NH2  (Formula  B),  or  anilin  oil,  which  is  the  basis  of 
all  anilin  stains  used  in  pathologic  and  bacteriologic  work.  In  this 
body  the  group  NH2,  which  is  attached  to  one  of  the  carbon  atoms, 
is  called  a  side-chain. 

According  to  the  hypothesis  of  Ehrlich,  the  chemical  substances 
of  cells  as  well  as  of  toxins  and  similar  bodies  contain  side-chains, 
which  by  uniting  in  the  animal  body  produce  both  poisonous  effects 
and  immunity.  How  this  is  accomplished  according  to  the  hypo- 
thesis will  be  shown.  The  chemical  formulae  of  the  live  cell  sub- 
stances, the  toxins  and  the  hypothetical  side-chains,  are  not  known. 
Therefore,  in  order  to  demonstrate  graphically  what  is  supposed 
to  happen,  figures  which  represent  the  matter  as  a  simple  physical 
arrangement  taking  place  between  geometrical  bodies  are  employed. 

Side-chains. — The  side-chain  theory  assumes  that  a  toxin  has  one 
side-chain  which  can  attach  itself  to  the  side-chain  of  a  cell.  After 
this  has  occurred  another  side-chain  of  the  toxin  fastens  itself  to  a 
second  side-chain  of  the  same  cell.  It  is  only  after  this  second  attach- 
ment has  taken  place  that  damage  is  done  to  the  cell.  In  other  words, 
the  first  side-chain  only  serves  to  anchor  the  toxin  to  the  cell,  and 
the  second  side-chain  of  the  toxin  produces  the  poisonous  effect. 

The  four  side-chains  in  this  mutual  process  between  the  cell  and 
the  toxin  have  received  special  names . 

Haptophore  Group. — The  side-chain  which  simply  attaches  the 
toxin  to  the  cell. 


TOXOIDS  75 

Haptophile  Group. — The  side-chain  of  the  cell  to  which  the  toxin 
becomes  attached. 

Toxophore  Group. — The  side-chain  of  the  toxin  which  has  the 
poisonous  effect. 

Toxophile  Group. — The  side  chain  of  the  cell  to  which  the  toxo- 
phore  group  of  the  toxin  becomes  attached,  and  by  which  attachment 
the  damage  is  done. 

Receptors. — Side-chains  of  the  cells  which  unite  with  the  side- 
chains  of  the  toxins  are  also  called  by  a  general  term,  the  cell  recep- 
tors. It  must  be  assumed  that  the  attachment  of  the  side-chains  of 
the  toxin  to  the  cell  receptors  destroys  the  latter,  at  least,  so  far 
as  their  character  and  existence  as  side-chains  are  concerned.  It  is 
a  general  observation  in  pathology  that  the  destruction  of  physiologic 
parts  is  always  followed  by  an  attempt  on  the  part  of  the  organism 
to  replace  what  has  been  lost.  So  if  any  liver  or  kidney  cells  have 
become  necrotic  the  organism  tries  to  replace  them  by  new  cells; 
this  is  called  a  regeneration.  Very  frequently  an  attempt  at  regen- 
eration leads  to  the  production  of  an  excess  of  that  which  had  been 
lost. 

The  side-chain  theory  assumes  that  whenever  cell  receptors  have 
been  made  useless  by  toxins  the  cell  not  only  replaces  the  lost  recep- 
tors but  that  it  produces  them  in  great  excess.  So  great  does  this 
excess  become  that  the  cell  body  cannot  retain  all  the  new  receptors 
or  new  side-chains,  and  it  must  expel  a  large  number  of  them  into 
the  blood  serum  and  other  juices  of  the  body.  If  an  animal  which 
possesses  a  large  number  of  such  receptors  in  its  blood  serum  becomes 
infected  a  second  time  by  the  same  toxin,  what  occurs?  Clearly, 
before  the  toxins  have  a  chance  to  attach  themselves  to  the  cells  they 
are  caught,  as  it  were,  by  the  free  floating  receptors  which  prevent 
them  reaching  the  cells  and  render  them  harmless.  Thus  the  animal 
is  protected  against  them.  A  serum  full  of  free  receptors  which  will 
catch  and  unite  with  the  toxins  is  called  an  immune  serum  or  an 
antitoxic  serum.  In  fact,  the  free  receptors  are  the  antitoxin  which 
unites  with  the  toxin  to  protect  the  cells. 

Toxoids. — It  has  been  stated  that  the  toxin  possesses  a  hapto- 
phore  and  a  toxophore  group.  This  can  be  shown  by  the  observation 
that  solutions  of  toxins  after  some  time  lose  much  or  all  of  their  poi- 
sonous properties,  and  yet  these  poisonless  non-toxic  toxins  may 
still  be  able  to  anchor  themselves  to  cells  and  cause  the  formation  of 
free  receptors.  In  other  words,  they  can  still  be  used  to  manufac- 
ture an  antitoxic  immune  serum  in  the  body  of  an  animal.  The 
toxin  has  lost  its  toxophore  side-chain  or  group,  but  it  still  possesses 
its  haptophore  side-chain  or  group.  Such  a  toxin  is  called  a  toxoid. 
Ehrlich  has  introduced  into  the  nomenclature  of  serum  investigation 
the  two  symbols  L0  and  L+.  The  letter  L  stands  for  the  term  lethal 
(deadly,  fatal).  The  former  symbol  L0  designates  a  toxin-antitoxin 
mixture  which  is  completely  neutralized,  and  therefore  will  not  kill 


76     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

an  animal.    While  the  latter  symbol  L+  designates  a  toxin-antitoxin 
mixture  which  contains  one  fatal  dose  in  excess  which  will  kill  the 


f 


FIG.  33 


u 


—B 


••E 


--F 


Graphic  representation  of  receptors  of  the  first  and  third  orders  and  of  complement  as  con- 
ceived by  Ehrlich:  A,  complement;  B,  intermediary  or  immune  body;  C,  cell  receptor;  D,  part 
of  cell;  E,  toxophorous  group  of  toxin;  F,  haptophorous  group.  (Park.) 


experimental  animal  within  a  few  days.  If  a  toxin  is  freshly  prepared 
and  its  minimum  fatal  dose  ascertained  it  will  be  found  that  a  cer- 
tain amount  of  antitoxin  is  required  to  neutralize  it  completely.  If 

100  fatal  doses  are  taken  it  will  require 
FlG-  34  100  protective  doses  of  the  antitoxin  to 

produce  a  completely  neutralized  mix- 
ture (L0).  However,  when  the  toxin 
gets  older  it  can  be  shown  that  instead 
of  100  protective  doses  only  about  80 
are  required  to  prevent  a  fatal  effect. 
In  the  case  of  the  fresh  toxin  L+  minus 
L0  is  equal  to  one  fatal  dose;  but  after 
the  toxin  has  gotten  older  L+  minus 
L0  is  equal  to  apparently  21  fatal  doses. 
The  reason  for  this  peculiar  behavior 
has  been  already  explained  above.  The 
toxin  that  is  part  of  it  has  lost  its 
toxophore  group,  but  has  retained  its 
haptophore  group,  and  with  it  its  com- 
bining power  toward  free  cell  receptors 
on  antitoxins.  The  toxin  has  been 
changed  into  a  toxoid. 


Receptors  of  the  second  order  are 
pictured  in  c.  Here  e  represents  the 
haptophore  group,  and  d  the  zymo- 
phore  group  of  the  receptor,  /  being 
the  food  molecule  with  which  this 
receptor  combines.  Such  receptors 
are  possessed  by  agglutinins  and 
precipitins.  It  is  to  be  noted  that 
the  zymophore  group  is  an  integral 
part  of  the  receptor.  (Park.) 


PLATE   II 

NO  HAEMOLYSIS, 


— Complement. 


Antigen  (Ay.) 

and 

Antibody  (Ab.) 

have  united  and 

have  fixed  the 

Complement. 


— Amboceptor. 


--Blood  corpuscle. 


HAEMOLYSIS 


—Complement. 


—  Amboceptor. 


Receptor  or  side  chain] 
of  blood  corpuscle. 


—Blood  corpuscle. 


Complete 

•  haemolytic 

System. 


HEMOLYSINS  77 

The  conditions  of  interaction  and  neutralization  of  antigen  and 
antibody  are  not  always  as  simple  as  in  the  case  of  toxins  and  anti- 
toxins. They  are  more  complicated  with  reference  to  other  anti- 
bodies, and  it  will  be  well  to  look  into  one  important  example  of  this 
kind. 

Hemolysins. — If  we  inject  into  the  body  of  a  guinea-pig  at  proper 
intervals  and  in  proper  doses  cholera  spirilla  the  animal  develops 
an  immune  serum  which  has  the  property  of  dissolving  cholera 
spirilla.  This  property  of  the  immune  serum  is  due,  as  stated  before, 
to  a  particular  kind  of  antibody  known  as  lysins.  If  we  inject  into 
a  rabbit  the  red  blood  corpuscles  of  a  sheep  the  former  will  develop 
an  immune  serum  which  has  the  property  of  dissolving  sheep's  cor- 
puscles. This  property  of  the  injected  or  sensitized  rabbit's  serum 
is  due  to  an  antibody  of  the  lysin  type  called  a  hemolysin.  It  consists, 
as  can  easily  be  shown,  of  two  bodies  which  can  be  separated  and 
studied  separately.  One  of  these  two  constituents  which  make  up 
the  complete  hemolysin  is  already  found  in  the  normal  serum  of 
most  animals;  the  other  constituent  antibody  is  only  found  in  the 
immune  serum  (the  serum  of  the  sensitized  rabbit)  or  at  least 
found  there  only  in  large  amount.  The  body  found  in  normal 
serum  is  called  the  complement,  and  the  other  the  immune  body  or 
amboceptor  (why  this  latter  name  is  used  will  be  explained  presently). 
The  complement  is  an  antibody  which  is  very  easily  destroyed  and 
cannot  stand  a  temperature  of  56°  C.  if  applied  to  a  serum  containing 
it  for  thirty  minutes.  Hence,  the  complement  is  said  to  be  thermo- 
labile.  The  amboceptor,  on  the  other  hand,  can  well  stand  heating 
for  thirty  minutes  at  56°  C.;  therefore,  it  is  said  to  be  thermostabile. 
If  an  immune  serum  containing  a  hemolysin  is  heated  for  thirty 
minutes  at  56°  C.,  destroying  the  complement,  but  not  the  amboceptor, 
it  is  said  to  be  inactivated,  because  it  now  cannot  bring  about  hemo- 
lysis.  If,  however,  some  normal  non-heated  serum  which,  as  stated, 
contains  the  complement  is  added  to  an  inactivated  serum  the  im- 
mune serum  is  again  reactivated  because  it  can  once  more  bring 
about  hemolysis  (solution  of  the  red  blood  corpuscles).  This  method 
of  inactivating  the  immune  serum  and  using  it  in  connection  with 
reactivating  normal  serum  makes  it  possible  to  study  the  ambo- 
ceptor and  the  complement  separately  and  to  learn  how  they  act,  how 
they  anchor  themselves  to  the  red  blood  corpuscles,  and  how,  by  so 
doing,  they  bring  about  hemolysis.  The  process  by  which  this 
occurs  is  the  following:  The  amboceptor  has  one  group,  or  side-chain, 
which  fastens  itself  to  a  receptor  of  the  red  blood  corpuscle,  and  a 
second  group,  or  side-chain,  by  which  it  attracts  and  anchors  to  itself 
the  complement.  Only  after  the  amboceptor  has  become  united  to 
both  the  complement  and  the  red  blood  corpuscle  can  the  solution 
of  the  latter  (hemolysis)  take  place.  The  union  of  (1)  red  blood 
corpuscle,  (2)  amboceptor,  and  (3)  complement  is  called  a  complete 
hemolytic  system  or  chain.  This  explains  why  the  immune  body  in 


78     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

hemolysins  has  been  called  amboceptor,  which  means,  literally  trans- 
lated, "double  catcher,"  or  "double  taker." 

Deviation  of  Complement. — To  a  solution  containing  a  hemolytic 
amboceptor  and  a  complement,  something  may  be  added  which  will 
unite  with  the  complement,  and  the  solution  will  then  loose  its 
property  of  dissolving  blood  corpuscles  (to  produce  hemolysis).  The 
complement  has  united  to  something  else,  and  can  no  longer  unite 
with  the  amboceptor,  a  complete  hemolytic  system  cannot  be  formed, 
and,  therefore,  hemolysis  cannot  take  place.  In  other  words,  comple- 
ment deviation  prevents  hemolysis. 

Complement  deviation  can  be  brought  about  by  several  means; 
for  instance,  by  the  use  of  anticomplements  or  by  the  presence  of 
an  antigen  uniting  with  its  antibody.  These  two,  when  united, 
generally  form  a  combination  which  will  anchor  to  itself  the  com- 
plement and  so  prevent  hemolysis.  Complement  deviation  has  been 
here  explained  somewhat  at  length,  because  it  has  become  a  most 
important  method  in  practical  serum  diagnosis.  The  principle  was 
discovered  by  Bordet  and  Gengou.  They  found  that  if  an  antigen 
be  permitted  to  unite  with  its  specific  immune  body  or  immune 
amboceptor,  something  is  formed  by  the  union  which  will  attract  to 
itself  the  complement  of  a  hemolysin,  so  that  the  latter  can  no  longer 
bring  about  a  solution  of  the  red  blood  corpuscles.  This  peculiar 
occurrence  or  phenomenon  may  be  used  to  detect  antibodies.  Sup- 
pose the  presence  of  certain  antibodies  is  suspected  in  the  blood 
serum  of  a  person  or  animal  they  can  be  detected  in  the  following 
manner:  Add  to  the  blood  serum  the  antigen  of  the  suspected 
antibodies  and  a  hemolytic  complement.  Later,  add  the  hemolytic 
amboceptor  and  some  red  blood  corpuscles.  If  solution  of  the  latter 
occurs  the  complement  is  not  deviated,  because  the  suspected  anti- 
body is  not  present,  and  could  not  unite  with  the  antigen,  and  could 
not,  therefore,  produce  complement  deviation.  If,  however,  there  is 
no  hemolysis  of  the  red  blood  corpuscles  it  clearly  shows  that  the 
suspected  antibody  is  present.  It  has  united  with  its  antigen  and 
they  in  their  union  have  attracted  the  complement,  which  is  no  longer 
present  in  the  free  state;  hence,  the  hemolytic  chain,  composed  of 
red  blood  corpuscles,  hemolytic  amboceptor,  and  hemolytic  comple- 
ment, cannot  be  formed  and  hemolysis  cannot  occur. 

The  Wassermann  Test. — Syphilis  in  man  is  often  very  difficult  to 
diagnosticate,  and  it  is  still  more  difficult  to  determine  whether  the 
disease  has  been  cured  or  whether  it  is  still  going  on  in  a  latent  form. 
The  phenomenon  of  complement  deviation  under  properly  arranged 
tests  has,  however,  furnished  a  means  of  an  almost  absolutely  infal- 
lible diagnosis.  Since  the  same  principle  may  be  applied  to  diseases 
of  domestic  animals,  and,  perhaps,  particularly  to  dourine  or  horse 
syphilis,  also  very  difficult  to  diagnosticate,  it  is  important  to  know  in 
detail  the  steps  of  the  serum  diagnosis  of  syphilis,  which  has  assumed 
such  great  importance  in  human  medical  practice.  The  author  has 


PLATE  III 


Positive.  Negative. 

Wassermann  Test. 


THE  WASSERMANN  TEST  79 

for  some  time  tried  to  find  a  chance  to  apply  his  test  to  dourine  of 
horses,  but,  unfortunately,  no  occasion  has  offered.  Very  probably 
the  test  could  be  made  in  an  identical  manner,  but  this  would,  of 
course,  have  to  be  tried  out  first  experimentally.  The  serum  test 
for  the  diagnosis  of  syphilis  was  devised  by  Wassermahn  and  his 
co-workers,  Neisser  and  Bruck.  It  is  generally  known  simply  as  the 
Wassermann  test. 

The   following  reagents   and  preliminary   steps   in   their  proper 
arrangement,  dilution,  etc.,  are  necessary: 

1.  The  red  blood  corpuscles  of  the  sheep  are  used  for  the  final 
hemolytic  tests.     They  must  be  washed  free  from  all  blood  serum. 
The  blood  is  obtained  either  from  the  jugular  vein  of  the  live  animal 
or  from  a  slaughter  house  at  the  time  when  the  animal  is  killed.    It 
is  best  to  have  the  blood  run  into  a  sterile  vessel,  though  this  is  by  no 
means  absolutely  necessary.    It  is  at  once  defibrinated  by  beating  it 
with  a  bundle  of  wires  or  glass  rods  or  by  shaking  it  with  some  frag- 
ments of  glass  or  glass  pearls.    After  the  fibrin  has  coagulated  there 
remains  an  intensely  red  fluid  containing  the  serum  and  the  cor- 
puscles.   Some  of  this  fluid  is  mixed  in  the  proportion  of  one  to  ten 
with  physiologic  salt  solution.    Two  centrifuge  tubes  are  filled  with 
the  mixture,  and  these  are  centrifuged  for  from  five  to  ten  minutes. 
There  is  now  formed  a  deposit  of  corpuscles  and  an  upper  layer  of 
clear  fluid.    The  latter  is  pipetted  off;  the  tubes  are  again  filled  with 
normal  salt  solution,  shaken  so  that  the  corpuscles  and  the  fluid  are 
mixed;  they  are  centrifuged  again  and  the  clear  fluid  is  pipetted 
off  once  more.    This  is  done  at  least  three  times.    Finally,  about  one 
part  of  the  washed  sheep's  corpuscles  is  mixed  with  nineteen  parts 
of  physiologic  salt  solution  and  shaken  until  an  emulsion  is  obtained. 
The  latter  is  called  the  5  per  cent,  emulsion  of  washed  sheep's  cor- 
puscles. 

2.  In  order  to  make  the  test  properly  it  is  necessary  to  have  a 
strong  hemolytic  amboceptor.      This   is  obtained   by  "sensitizing  a 
rabbit  against  sheep's  corpuscles"  in  the  following  manner:     The 
abdominal  region  of  the  rabbit  is  shaved  and  cleansed  antiseptically. 
The  animal  is  then  held  up  by  two  assistants  in  such  manner  that 
its  head  hangs  down  and  the  hind  legs,  which  are  spread  out,  point 
upward.      In  this  position  the  rabbit's  intestines   fall  toward  the 
diaphragm  and  there  is  little  danger  of  injuring  them  in  the  next 
step,  which  consists  in  injecting  into  its  peritoneal  cavity  about  10  c.c. 
of  a  5  per  cent,  physiologic  salt  emulsion  of  washed  sheep's   cor- 
puscles (this  must  have  been  freshly  prepared  under  aseptic  precau- 
tions and  the  injection  must  be  made  with  a  sterile  syringe).     This 
procedure  is  repeated  three  or  four  times  or  oftener  at  intervals  of 
about  ten  days.     The  blood  serum  of  a  rabbit  so  treated  will  have, 
as  a  rule,  a  very  strong  hemolytic  power  toward  sheep's  corpuscles. 
Since  the  serum  is  sometimes  deficient  in  spite  of  the  injection  treat- 
ment, it  is  well  to  draw  some  blood  from  an  ear  vein  and  test  it 


80     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

before  all  of  the  rabbit's  serum  is  collected.  If  the  preliminary  trial 
shows  a  strong  hemolytic  power,  the  rabbit  is  killed  by  cutting 
both  carotid  arteries  and  allowing  the  blood  to  flow  into  a  sterile 
cylindrical  vessel.  The  blood  is  allowed  to  coagulate,  and  is  then 
placed  on  ice  for  a  number  of  hours,  after  which  the  serum  is 
removed  from  the  coagulum.  If  the  serum  is  not  perfectly  clear  and 
still  contains  some  red  blood  corpuscles,  it  must  be  centrifuged. 
The  clear  serum  is  finally  pipetted  off.  This  is  next  heated  on  a 
water  bath  at  56°  C.  for  thirty  minutes.  By  so  inactivating  the 
serum,  we  destroy,  as  explained  before,  the  hemolytic  complement, 
so  that  the  rabbit's  serum  will  now  alone  not  hemolyze  sheep's  cor- 
puscles. 

3.  The  complement   necessary  to  produce  hemolysis   is  obtained 
in  the  blood  serum  of  the  guinea-pig.     An  animal  of  this  kind  is 
bled  to  death  by  severing  the  carotid  arteries.     The  blood  is  col- 
lected at  once  in  some   centrifuge  tubes,  and  after  coagulation  is 
centrifuged  and  the  serum  pipetted  off  from  the  clot.     The  comple- 
ment in  the  guinea-pig's  serum  is  very  easily  destroyed,  by  simply 
allowing  the  serum  to  stand  at  room  temperature.    If  the  serum  cannot 
be  used  at  once  it  must  be  placed  in  the  refrigerator,  and  if  it  is  to 
be  kept  for  more  than  twenty-four  hours,  it  must  be  frozen  hard  in  a 
mixture  of  salt  and  ice  and  kept  in  a  thermo  bottle  in  a  freezing  mixture. 

4.  The  rabbit's  inactivated  serum  is  now  tested  in  the  following 
manner : 

(a)  Take  a  number  of  test  tubes  and  place  into  each  ten  drops  of 
a  5  per  cent,  physiologic  salt  solution  emulsion  of  washed  sheep's 
corpuscles. 

(6)  Add  to  them  one  drop  of  the  guinea-pig's  serum  and  nine  drops 
of  salt  solution. 

(c)  Now  add  to  the  different  test-tubes  varying  amounts  of  the 
inactivated  rabbit's  blood  serum  plus  enough  salt  solution  to  make 
ten  drops,  say  as  follows: 

To  tube  No.  1,  add  one  drop. 

To  tube  No.  2,  add  one-half  drop. 

To  tube  No.  3,  add  one-fourth  drop. 

To  tube  No.  4,  add  one  eighth  drop. 

To  tube  No.  5,  add  one-twelfth  drop. 

To  tube  No.  6,  add  one-sixteenth  drop. 

These  additions  have  to  be  made  exactly  by  previously  diluting  some 
of  the  inactivated  rabbit's  serum  accurately;  for  instance,  dilution 
No.  6  would  be  made  by  adding  to  one  drop  of  serum  fifteen  drops 
of  salt  solution  and  taking  of  this  dilution  one  drop  plus  nine  drops 
of  salt  solution  to  test-tube  No.  6. 

(d)  Finally,  add  to  each  tube  twenty  more  drops  of  the  salt  solution, 
so  that  each  tube  contains  exactly  fifty  drops.    Shake  them  well  and 
place  them  all  for  thirty  minutes  into  the  incubator.     Examine  after 
they  have  been  incubated  one-half  hour. 


THE  WASSERMANN  TEST.  81 

Suppose  that  at  this  time  all  the  tubes  except  No.  6  show  complete 
hemolysis.  This  one  shows  only  partial  hemolysis.  This  means 
that  one-twelfth  of  a  drop  of  the  inactivated  blood  serum  of  the  rabbit 
will  be  sufficient  for  complete  hemolysis  with  the  quantities  used  in 
the  test.  It  is  customary  to  use  in  the  final  determining  tests  about 
three  times  the  minimum  amount  of  rabbit's  serum  which  will  bring 
about  complete  hemolysis.  Hence,  if  the  final  determining  tests  are 
arranged  in  the  same  proportion  as  the  tests  made  to  ascertain  the 
titre  (or  strength)  of  the  rabbit's  serum,  one-quarter  of  a  drop  of 
this  serum  would  be  used. 

5.  It  is  now  necessary  to   make    another   test  by  adding  to  ten 
drops  of  a  5  per  cent,  washed  sheep's  corpuscles  emulsion,  diluted 
with  forty  more  drops  of  salt  solution,  one  drop  of  rabbit's  serum  and 
placing  this  tube  into  the  incubator  for  several  hours.    At  the  end  of 
this  time  there  must  not  be  any  hemolysis  at  all.     This  will  show 
that  the  rabbit's  serum  has  been  inactivated  properly,  and  that  it 
contains  no  more  complement,  the  latter  has  been  entirely  destroyed 
and  cannot  interfere  in  the  final  test.    The  inactivated  rabbit's  serum 
must  be  kept  in  the  refrigerater,  where  it  will  keep  for  several  months. 

6.  Another  necessary  reagent  is  an  extract  which  will    contain 
the  antigen  of  the  suspected  syphilitic  antibodies  (or  syphilitic  ambo- 
ceptor)  the  presence  or  absence  of  which  in  the  blood  serum  to  be  exam- 
ined is  to  be  determined  by  the  test.    This  antigen  cannot  be  prepared 
from  a  pure  culture  of  the  organism  causing  syphilis,  because  so  far 
it  has  been  impossible  to  obtain  it  in  pure  culture.     It  can  be  pre- 
pared, however,  from  the  liver  of  a  newborn  child  dead  from  con- 
genital syphilis.    This  liver  is  cut  up  into  small  pieces  and  one  part 
by  weight  of  liver  is  rubbed  up  very  thoroughly  with  five  parts  of 
absolute  alcohol.     The  mixture  is  then  shaken  in  a  shake  machine 
for  twelve  to  twenty-four  hours,  allowed  to  stand,  then  filtered  and 
the  clear  filtrate  known  as  the  alcoholic  antigen  extract  is  ready  for 
use. 

7.  Complement  deviation  in  the  case  of  syphilis  is  not  an  abso- 
lutely strictly  specific  reaction,  which  means  that  the  syphilitic  antigen 
extract  can  be  replaced  by  some  other  preparations  which  will  do  the 
same  work,  and  unite  with  the  syphilitic  antibodies  and  deviate  the 
complement.     The  substitute  most  commonly  used  in  place  of  the 
luetic  antigen  is  an  alcoholic  extract  of  guinea-pig's  heart,  which  is 
prepared  as  follows:    After  the  animal  is  dead  the  heart  is  removed, 
washed  in  physiologic  salt  solution,  dried  between  filter  papers,  and 
weighed.     The  heart  is  then  cut  up,  and  the  parts  dropped  into  a 
mortar  which  contains  washed  sterile  quartz  sand;  add  fifty  times  the 
weight  of  the  heart  of  95  per  cent,  alcohol  and  triturate  with  the  alcohol 
and  sand  until  the  heart  has  been  very  finely  divided.   The  entire  mix- 
ture is  next  removed  from  the  mortar  to  a  flask  or  other  suitable  glass 
vessel  and  heated  on  a  water  bath  for  three  to  four  hours  at  60°  C. 
After  cooling,  the  evaporated  alcohol  must  be  made  up  to  the  original 


82     ANTIBODIES,  IMMUNITY,   WASSERMANN  SERUM  TEST 

bulk.  It  is  now  filtered  off  from  the  sand,  etc.,  and  the  clear  alcoholic 
extract,  placed  in  a  tightly  glass-stoppered  bottle,  is  ready  for  use  at 
any  time.  This  alcoholic  extract  of  guinea-pig's  heart  keeps  for 
many  months  at  room  temperature,  provided  it  is  protected  against 
evaporation  and  contamination. 

The  blood  sera  used  for  the  tests  are  the  serum  from  the  patient 
to  be  examined,  the  serum  from  a  healthy  person  and  a  serum  from 
one  who  is  in  the  early  stages  of  syphilis.  The  two  latter  sera  are, 
of  course,  used  as  controls  in  connection  with  tests  of  the  patient's 
serum.  Blood  is  obtained  from  the  three  persons  with  a  sterile,  all- 
glass,  large  hypodermic  syringe,  the  barrel  of  which  will  hold  from 
five  to  ten  cubic  centimeters  of  blood.  After  the  latter  has  been 
drawn  under  all  aseptic  precautions  from  a  vein  of  the  arm,  it  is  at 
once  placed  in  a  centrifuge  tube.  After  coagulation  the  clot  is  loosened 
from  the  walls  of  the  tube  with  a  platinum  wire,  the  tube  is  cen- 
trifuged,  and  the  serum  pipetted  off.  If  not  clear,  it  is  centrifuged  a 
second  time.  After  the  three  clear  sera  have  been  obtained  they  are 
heated  for  thirty  minutes"  at  56°  C.  on  a  water  bath.  This  inactivating 
is  done  to  destroy  the  complement  present,  but  does  not  interfere  with 
the  syphilitic  antibody  or  syphilitic  amboceptor. 

8.  Everything  is  now  ready  to  make  the  test,  in  the  following 
dilutions.  The  alcoholic  antigen  extract  is  used  in  a  dilution  of  two 
in  ten,  with  physiologic  salt  solution.  The  rabbit's  inactivated  serum 
which  contains  the  hemolytic  amboceptor  is  used  in  a  dilution  of  one 
drop  in  forty  or  one-quarter  of  a  drop  in  ten  of  the  salt  solution.  The 
reagents  are  now  employed  as  follows : 

(a)  Alcoholic  extract  of  antigen  (dilution  two  in  ten). 

(b)  Three  inactivated  sera,  one  from  the  patient,  two  controls. 

(c)  The  fresh  unheated  serum  from  the  guinea-pig  containing  the 
hemolytic  complement. 

(d)  The  inactivated  rabbit's  serum  (dilution  one  in  forty)  contain- 
ing the  hemolytic  amboceptor. 

(e)  The  5  per  cent,  washed  sheep's  corpuscles  salt  solution  suspen- 
sion, containing  the  sheep's  corpuscles,  on  which  hemolysis  will  be 
tried. 

The  test  is  now  made  as  follows: 

Prepare  six  test-tubes  and  label  as  follows :  Patient  No.  1 ;  Patient 
No.  2;  Positive  control,  No.  1 ;  Positive  control,  No.  2;  Negative  con- 
trol, No.  1;  Negative  control,  No.  2.  Also  label  the  tubes  in  this 
order  from  No.  1  to  No.  6,  consecutively.  Now  add  to  each  tube  as 
follows:  * 

To  No.  1  (patient  No.  1),  10  drops  alcoholic  antigen  extract,  plus 
2  drops  of  patient's  serum,  plus  8  drops  of  salt  solution,  plus  1  drop 
of  guinea-pig's  serum,  plus  9  drops  of  salt  solution. 

To  No.  2  (patient  No.  2),  10  drops  of  salt  solution,  plus  2  drops  of 
patient's  serum,  plus  8  drops  of  salt  solution,  plus  1  drop  of  guinea- 
pig's  serum,  plus  9  drops  of  salt  solution. 


THE  WASSERMANN  TEST  83 

To  No.  3  (positive  control  No.  1),  10  drops  of  alcoholic  antigen 
extract,  plus  2  drops  of  serum  of  syphilitic,  plus  8  drops  of  salt  solu- 
tion, plus  1  drop  of  guinea-pig's  serum,  plus  9  drops  of  salt  solution. 

To  No.  4  (positive  control  No.  2),  10  drops  of  salt  solution,  plus 
2  drops  of  syphilitic  serum,  plus  8  drops  of  salt  solution,  plus  1  drop 
of  guinea-pig's  serum,  plus  9  drops  of  salt  solution. 

To  No.  5  (negative  control  No.  1),  10  drops  alcoholic  antigen 
extract,  plus  2  drops  of  healthy  person's  serum,  plus  8  drops  of  salt 
solution,  plus  1  drop  of  guinea-pig's  serum,  plus  9  drops  of  salt 
solution. 

To  No.  6  (negative  control  No.  2),  10  drops  of  salt  solution,  plus 
2  drops  of  healthy  persons  serum,  plus  8  drops  of  salt  solution, 
plus  1  drop  of  guinea-pig's  serum,  plus  9  drops  of  salt  solution. 

We  have  now  three  sets  of  tubes,  each  set  containing  the  serum  of 
a  different  person,  and  in  each  set  one  tube  with  alcoholic  antigen 
extract  and  one  set  without  this  extract,  its  place  being  taken  by 
physiologic  salt  solution.  There  must  be  another  set  of  controls  pre- 
pared from  the  sera  to  find  out  whether  the  extract  is  of  the  proper 
strength.  In  these  controls  the  alcoholic  extract  is  present  in  the  fol- 
lowing proportions :  1  to  10,  2  to  10,  4  to  10, 6  to  10.  The  extract  should 
be  of  such  strength  that  1  to  10  is  strong  enough  to  inhibit  hemolysis 
with  a  serum  from  a  case  of  syphilis;  4  to  10  should  be  of  such  a  strength 
that  the  presence  of  a  syphilitic  antibody  is  necessary  to  prevent 
haemolysis,  and  6  to  10  should  be  strong  enough  to  prevent  it  alone. 
These  additional  tests  need  not  be  made  every  time,  but  just  often 
enough,  so  that  the  alcoholic  antigen  extract  is  always  kept  under 
control.  Another  point  about  this  extract  is  the  following:  Since 
it  contains  in  the  proper  dilution  about  25  per  cent,  alcohol,  its 
drops  are  smaller  than  the  drops  of  the  purely  watery  fluids  used, 
hence  it  is  necessary  to  use  a  special  dropper  for  the  alcoholic  extract 
which  has  been  tested  out,  or  if  the  same  dropper  or  pipette  is  used, 
it  is  necessary  to  take  from  twelve  to  thirteen  drops  of  the  dilute 
alcohoic  extract  for  each  ten  drops  of  the  watery  solutions.  All 
reagents  must  be  used  so  that  they  represent  in  the  salt  solutions 
equal  amounts,  namely,  ten  drops. 

After  the  test-tubes  have  been  prepared  they  are  placed  in  the 
incubator  for  thirty  minutes.  If  there  is  present  in  any  test-tube  a 
combination  of  antigen  and  syphilitic  antibody,  these  will,  during 
the  half-hour  in  the  incubator,  unite  and  attract  and  fix  to  themselves 
the  complement  which  is  present  in  each  tube. 

After  thirty  minutes  the  six  test-tubes  and  the  other  controls  which 
may  have  been  made  are  removed  from  the  incubator  and  to  each 
one  is  added : 

10  drops  of  the  diluted  inactivated  rabbit's  blood  serum  contain- 
ing the  hemolytic  amboceptor. 

10  drops  of  the  5  per  cent,  washed  sheep's  corpuscles  salt  solu- 
tion suspension. 


84      ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

The  tubes  are  well  shaken  and  replaced  in  the  incubator  for  one  to 
one  and  one-half  hours.  They  are  then  taken  out  and  the  result  may 
be  recognized  at  once,  or,  better,  the  tubes  are  placed  overnight  in 
the  refrigerator.  This  will  enable  all  undissolved  corpuscles  to  sink 
to  the  bottom  of  the  tubes  and  a  very  characteristic  unmistakable 
picture  is  formed.  Hemolyzed  tubes  show  a  uniformly  transparent 
red  stained  fluid.  Non-hemolyzed  tubes  show  a  sediment  of  red  blood 
corpuscles  at  the  bottom  and  a  perfectly  clear  supernatant  salt  solu- 
tion on  top.  The  result  of  the  test  in  case  the  suspected  patient  has 
syphilis  will  be: 

Tube  No.  1  (patient  No.  1),  no  hemolysis. 

Tube  No.  2  (patient  No.  2),  hemolysis. 

Tube  No.  3  (positive  control  No.  1),  no  hemolysis. 

Tube  No.  4  (positive  control  No.  2),  hemolysis. 

Tube  No.  5  (negative  control  No.  I),  hemolysis. 

Tube  No.  6  (negative  control  No.  2),  hemolysis. 

The  result  is  explained  as  follows:  The  patient  has  syphilis,  hence 
in  tube  No.  1  the  antigen  (alcoholic  extract)  and  the  syphilitic  anti- 
bodies united;  they  deviated  the  complement  and  hemolysis  could 
not  take  place.  The  same  conditions  prevail  as  to  tube  No.  3;  the 
blood  came  from  a  person  known  to  have  syphilis.  No.  5  con- 
tained the  blood  of  a  healthy  person,  hence  the  antigen  (alcoholic 
extract)  could  not  unite  with  syphilitic  antibodies,  since  none  were 
present;  consequently,  hemolysis  took  place.  In  tubes  Nos.  2,  4,  and 
6  no  alcoholic  antigen  extract  was  added;  hence,  even  if  syphilitic 
antibodes  are  present,  as  in  tubes  Nos.  2  and  4,  they  had  no 
antigen  to  unite  with,  and  hence  could  not  deviate  the  complement, 
and  hemolysis  took  place. 

If  the  patient  does  not  have  syphilis,  hemolysis  will,  of  course, 
take  place  in  Tube  No.  1;  since  there  are  no  syphilitic  antibodies 
present  they  cannot  unite  with  the  antigen,  and  the  complement 
will  not  be  deviated. 

Anaphylaxis  and  Hypersusceptibility. — A  very  peculiar  occurrence, 
not  as  yet  fully  understood,  has  been  observed  and  studied  experi- 
mentally by  Arthus,  Theobald  Smith,  Rosenau  and  Anderson,  and 
others.  If  an  animal  receives  a  very  small  hypodermic  or  intra- 
peritoneal  injection  of  an  alien  or  heterologous  blood  serum,  that  is, 
a  blood  serum  from  an  animal  of  a  different  species,  and  after  the 
lapse  of  about  three  weeks  a  larger  dose  (for  instance,  several  cubic 
centimeters)  of  the  same  blood  serum,  a  very  grave  condition  fre- 
quently develops.  An  animal  so  treated  may  show  difficulty  in  res- 
piration, rapid  pulse,  convulsions,  and  death.  There  has  evidently 
been  established  in  the  animal  in  consequence  of  the  first  small  dose 
of  the  alien  serum  a  hypersusceptibility  to  this  serum.  However,  if 
animals  showing  this  complex,  of  symptoms  do  not  die  they  rapidly 
get  over  the  attack  and  are  soon  well  again.  Anaphylaxis  can  be 
produced  both  actively  by  injecting  into  a  guinea-pig  an  alien  serum 


QUESTIONS  85 

(for  example  that  of  the  horse)  and  passively  by  taking  the  serum 
of  the  same  guinea-pig,  after  two  or  three  weeks,  and  injecting  it 
into  another  guinea-pig,  producing  in  it  the  same  anaphylaxis  or 
hypersusceptibility  against  the  alien  serum. 

Sudden  Unexplained  Loss  in  Antitoxic  Value. — Another  occasional 
peculiar  occurrence  difficult  to  explain  with  our  present  knowledge  of 
the  details  of  the  processes  of  immunity  is  the  following: 

A  horse  may  have  been  highly  immunized  (hyperimmunized) 
against  tetanus  toxin.  The  blood  serum  of  this  horse  shows  an  enor- 
mous amount  of  antitoxin — in  other  words,  it  shows  in  each  cubic 
centimeter  a  high  figure  of  immunity  units.  The  animal  receives 
another  large  dose  of  tetanus  toxins.  This  ordinarily  would  cause 
the  antitoxic  value  of  its  serum  to  rise  still  higher,  or,  at  least,  if  the 
limit  has  been  reached  to  remain  stationary.  Instead  of  this  the 
antitoxic  value  sinks  enormously,  the  toxins  are  not  properly  neu- 
tralized, and  the  horse  gets  very  sick  and  may  even  die.  There  is 
no  satisfactory  explanation  for  this  peculiar  occurrence  which  appar- 
ently opposes  all  dicta  of  the  theories  of  immunity. 

QUESTIONS. 

1.  What  is  an  antitoxin?    What  effect  has  it  upon  a  toxin? 

2.  How  can  this  effect  be  demonstrated? 

3.  What  happens  if  we  inject  cholera  spirilla  into  the  peritoneal  cavity  of 
an  animal  not  very  susceptible  to  them? 

4.  What  is  an  agglutinin?    What  is  a  lysin? 

5.  What  properties  does  a  rabbit's  blood  serum  acquire  if  we  inject  into  this 
animal  human  blood  serum  several  times  at  intervals  ? 

6.  What  is  a  precipitin  ?    Describe  the  procedure  necessary  to  produce  pre- 
cipitins  for  horse's  blood  serum  in  the  body  of  a  rabbit. 

7.  What  practical  use  has  been  made  of  the  precipitin  test  in  forensic  medicine  ? 

8.  Give  a  definition  of  the  term  antibodies. 

9.  What  is  an  antigen? 

10.  What  are  the  antibodies  against  tetanus  and  diphtheria  toxins?    How 
prepared  and  obtained  ? 

11.  What  is  the  paraenteral  method  of  producing  antibodies? 

12.  What  is  meant  by  virulency;  what  by  attenuation?    How  can  virulent 
bacteria  or  their  toxins  be  attenuated  ? 

13.  What  is  meant  by  a  vaccine? 

14.  What  is  an  immune  serum? 

15.  What  is  meant  by  the  term  immunity? 

16.  What   is   congenital   natural   immunity?     What   is   naturally   acquired 
immunity  ? 

17.  What  is  artificial  immunity?    What  is  the  difference  between  active  and 
passive  immunity  ? 

18.  What  is  the  simultaneous  method  of    immunizing  an  animal?      Name 
some  diseases  against  which  this  method  is  used. 

19.  What  circumstances  influence  the  union  between  toxin  and  antitoxin? 

20.  What  is  the  benzole  ring  of  Kekule  ? 

21.  What  does  the  term  side-chain  mean  as  used  in  organic  chemistry? 

22.  Explain  the  terms:   haptophore,  haptophile,  toxophore,  toxophile,  side- 
chains. 

23.  Explain  the  interaction  of  these  side-chains  toward  each  other. 

24.  What  generally  happens  if  tissue  elements-are  destroyed  ? 

25.  What  is  a  cell  receptor? 

26.  What   happens  according  to  the  side-chain  theory  if   cell  receptors  are 
destroyed  ? 


86     ANTIBODIES,  IMMUNITY,  WASSERMANN  SERUM  TEST 

27.  What  are  the  names  given  to  the  free  side-chains  contained  in  the  blood 
serum?     What  is  their  relation  to  toxins  liberated  in  the  body  of  an  infected 
animal? 

28.  What  is  a  toxoid  ?    Under  what  conditions  is  it  formed  ? 

29.  What  is  a  hemolysin?     Is  it  a  simple  or  a  compound  antibody?     What 
enters  into  its  formation? 

30.  What  is  the  meaning  of   thermolabile  and  thermostabile  ?     What  does 
inactivating  mean  ? 

31.  What  is  an  amboceptor?    Why  so  named? 

32.  How  can  a  previously  inactivated  serum  be  reactivated  ? 

33.  What  is  a  complete  hemolytic  system  or  chain? 

34.  How  can  the  hemolytic  complement  be  deviated  or  fixed  ? 

35.  How  can  a  rabbit  be  sensitized  so  that  its  blood  serum  will  bring  about 
hemolysis  of  sheep's  corpuscles? 

36.  Describe  the  method  of  washing  sheep's  corpuscles. 

37.  How  is  the  hemolytic  complement  for  tests  in  hemolysis  generally  obtained  ? 

38.  How  must  this  complement,  which  is  easily  destroyed,  be  preserved  ? 

39.  Describe  the  method  of  inactivating  sensitized  rabbit's  serum. 

40.  How  are  the  clear  sera  from  animals  or  man  obtained?    How  are  they 
treated  before  use  ? 

41.  In  what  proportions  and  dilutions  are  the  reagents  in  a  test  for  complement 
deviation  used? 

42.  How  is  the  antigen  extract  in  Wassermann's    serum  test  for  syphilis 
prepared  ? 

43.  Describe  in  detail  the  steps  of  the  Wassermann  test. 


CHAPTER    VIII. 

METHODS  OF  OBSERVING  BACTERIA— THE  USE  OF  THE 
MICROSCOPE  AND  ACCESSORIES. 

Cultures. — It  is  absolutely  necessary  in  the  study  of  pathogenic 
bacteria  to  obtain  each  definite  species  free  from  any  other  live 
organisms.  When  such  a  preparation  is  successfully  made  whether 
of  a  pathogenic  or  other  bacterium  it  is  called  a  pure  culture.  It  is 
generally  composed  of  small  discrete  masses  called  colonies,  and  these 
in  turn  are  formed  by  innumerable  individual  bacilli  which  in  their 
entity  make  up  the  pure  culture.  When  bacteria  have  grown  abund- 
antly on  a  properly  prepared  artificial  culture  soil  they  often  appear 
as  one  continuous  mass  of  the  growth  and  the  individual  colonies  are 
no  longer  distinguishable.  They  were  present  very  early  in  the  course 
of  the  development,  but  have  become  confluent.  If  the  bacterium 
has  originally  been  inoculated  into  the  culture  soil  in  a  very  dilute 
form  individual  colonies  can  always  be  seen. 

Microscopic  Study. — Several  of  the  properties  and  characteristics 
of  pathogenic  bacteria  in  pure  cultures  can  be  studied  with  the  naked 
eye,  such  as  the  varying  degrees  of  moisture  or  dryness,  the  smooth 
or  granular  surface  of  the  growth,  its  color,  its  power  to  liquefy  certain 
culture  media,  etc.  In  order  to  observe  the  individual  bacterium, 
however,  a  microscope  is  required  with  one  low  and  one  high  power 
lens,  and  an  especially  powerful  illuminating  apparatus.  The  prepar- 
ation of  pure  cultures  is  much  more  difficult  and  time-consuming 
than  the  microscopic  study  of  bacteria,  after  they  are  once  obtained 
in  a  pure  state;  hence,  the  student  should  first  familiarize  himself 
with  the  use  of  the  microscope  in  observing  bacteria  in  the  stained 
and  unstained  condition.  This  study  will,  therefore,  be  considered 
prior  to  the  subject  of  culture  media,  pure  cultures,  and  the  sterili- 
zation methods  necessary  to  obtain  them. 

Source  of  Light. — In  the  microscopic  study  of  bacteria,  natural, 
reflected,  diffuse  light  is  employed  during  the  day,  best  obtained  at 
a  window  with  a  northern  exposure.  When  artificial  light  is  used, 
an  ordinary  coal-oil  lamp  or  gas  burner  will  serve  the  purpose,  an 
Argand  burner  is  better,  and  a  Welsbach  light  is  best  of  all.  An 
Edison  incandescent  lamp  is  not  a  good  light,  and  if  used  at  all 
should  have  a  bulb  of  frosted  glass,  to  prevent  the  incandescent  film 
from  disturbing  the  field  of  vision. 

The  Microscope. — The  modern  microscope  consists  of  a  stand 
forming  the  foot  or  base  and  carrying  the  stage  and  a  brass  tube 


88  METHODS  OF  OBSERVING  BACTERIA 

containing  the  optical  apparatus,  which  can  be  moved  up  and  down 
on  a  supporting  arm  from  an  upright  pillar  of  the  stand.  The  raising 
and  lowering  of  this  tube  is  accomplished  by  a  rack  and  pinion, 
worked  by  a  large  screw-head  on  either  side.  This  arrangement  is 
called  the  coarse  adjustment.  In  the  outer  tube  is  an  inner  tube 
which  can  be  drawn  out  by  hand.  This  inner  tube  is  graduated  in 
millimeters,  and  if  the  lower  end  of  the  outer  tube  is  provided  with 
a  so-called  revolver  or  nosepiece  the  inner  tube  should  be  drawn 
out  to  the  mark  16  cm.  or  160  mm.  The  microscope  will  then  be 
so  adjusted  that  the  distance  from  the  upper  lens  of  the  eyepiece  to 
the  objective  is  170  mm.  (170  millimeters  =  17  centimeters).  This 
is  the  distance  to  which  the  higher  power  objectives  are  corrected 
and  at  which  they  work  best,  giving  the  clearest  picture  of  the  object 
under  observation. 

There  is,  in  addition  to  the  coarse  adjustment,  the  so-called  fine 
adjustment  worked  by  a  micrometer  screw,  placed  either  on  top  or 
at  the  sides  of  the  centre  pillar  of  the  instrument.  This  raises  and 
lowers  the  draw  tube  only  very  slightly  so  that  it  can  be  adjusted 
to  the  one-hundredth  part  of  a  millimeter. 

Condenser. — One  of  the  most  important  parts  of  the  instrument  is 
the  substage  or  Abbe  condenser,1  or  illuminating  apparatus.  This 
is  attached  below  the  central  opening  of  the  microscope  stage  in 
such  a  manner  that  it  has  considerable  movement  in  a  vertial  plane, 
allowing  the  upper  lens  of  the  condenser  to  be  on  a  level  with  the 
stage  when  desired.  The  importance  of  this  vertical  movement  will 
be  seen  presently.  The  substage  condenser  consists  of  three  parts, 
namely,  a  reflector,  an  iris  diaphragm  and  the  condenser  lens  proper. 

The  reflector  is  a  circular  disk  suspended  so  that  it  can  revolve 
around  the  median  axis  of  the  equatorial  plane,  and  has  two  sur- 
faces, a  plane  mirror  and  a  concave  mirror.  The  condenser  proper, 
which  forms  the  upper  part  of  the  illuminating  apparatus,  consists 
of  a  system  of  lenses.  Between  the  reflector  and  the  condenser  lens 
is  an  iris  diaphragm.  The  word  diaphragm  means  a  partition  wall, 
and  in  the  modern  microscope  this  wall  between  the  reflector  and 
the  condenser  opens  and  closes  in  a  concentric  manner  like  the  iris 
of  the  eye,  hence  the  name.  Those  who  have  done  photographic  work 
with  the  camera  understand  the  workings  of  the  iris  diaphragm;  it 
can  be  adjusted  so  that  it  forms  a  large  opening,  admitting  a  power- 
ful bundle  or  pencil  of  rays  of  light,  or  it  can  be  closed  through  all  the 
intermediate  stages  to  a  pinhole  opening  admitting  very  little  light. 
Some  of  the  modern  microscopes  possess  a  second  iris  diaphragm 
placed  above  the  substage  condenser  lens.  This  second  diaphragm 
is  used  in  place  of  the  lower  one  when  the  condenser  lens  has  been 
removed.  When  instruments  are  equipped  with  Abbe  condensers 
the  plane  mirror  of  the  reflector  should  be  used  and  not  the  concave 
mirror. 

1  Called  Abbe  condenser  because  invented  and  perfected  by  Professor  Abbe,  of  Jena. 


IMAGE  89 

Objectives  and  Eyepieces. — The  most  important  part  of  the  modern 
compound  microscope  is  the  objective  and  next  to  it  the  eyepiece. 
Upon  these,  particularly  the  former,  depends  the  clearness  of  definition 
and  details  of  the  image  obtained. 

Refraction. — The  natural  law  upon  which  the  whole  construction 
of  the  optical  parts  of  the  microscope  (objectives  and  eyepieces) 
depends  is  that  rays  of  light  are  deviated  from  their  course  when  they 
travel  from  a  transparent  medium  of  a  certain  density  into  a  trans- 
parent medium  of  a  different  density.  This  deviation  from  their 
path,  which  occurs  according  to  very  definite  mathematical  rules, 
is  called  refraction.  Objectives  and  eyepieces  can  be  so  arranged 
that  the  light  after  being  refracted  forms  an  enlarged  or  magnified 
image  in  the  eye  of  the  observer.  .  Objectives  are  of  low,  of  medium, 
and  of  high  magnification. 

Aberration. — High-power  objectives  or  lenses  must  be  corrected 
to  overcome  two  sources  of  defects  which  interfere  with  the  clearness 
of  the  image.  White  light  is  composed  of  a  number  of  component 
colors  (the  colors  of  the  spectrum  or  rainbow)  and  the  rays  of  dif- 
ferent colors  are  refracted  in  a  different  manner  by  refractive  media. 
Hence,  they  furnish  an  indistinct  picture,  not  true  in  color,  and  which 
is  confused  by  the  appearance  of  color  rings.  This  defect  of  high- 
power  lenses  is  called  their  chromatic  aberration.  The  other  defect 
is  due  to  the  very  strong  curvature  and  very  small  radius.  A  lens  so 
constructed  will  refract  the  rays  at  its  periphery  (margin)  differently 
from  the  other  rays,  forming  indistinct  pictures.  This  defect  is  called 
its  spherical  aberration.  Both  the  spherical  and  the  chromatic 
aberration  can  be  corrected  by  a  combination  of  lenses  made  up  of 
glasses  with  a  difference  in  their  refractive  index.  But  the  more  com- 
plete the  correction  the  more  difficult  and  delicate  is  the  construc- 
tion of  these  lenses,  and,  therefore,  the  greater  the  cost. 

Focus. — Every  microscopic  objective  is  a  system  of  lenses  which 
acts  as  a  convex  lens,  and  parallel  rays  of  light  passing  through  it  are 
refracted  so  that  they  meet  in  a  single  point  called  its  main  or  prin- 
cipal focus.  The  distance  of  this  point  or  focus  from  the  central 
point  of  the  lens  is  called  its  focal  distance.  In  microscopic  objec- 
tives the  higher  the  magnification  the  shorter  will  be  the  focal  dis- 
tance, and  the  lower  the  magnification  the  longer  this  distance. 

Image. — If  an  illuminated  object  is  placed  somewhere  between 
the  single  and  the  double  focal  distance  of  a  convex  lens  there  is 
formed  on  the  other  side  of  it  an  enlarged  or  magnified  real  image 
of  the  object.  In  the  use  of  the  compound  microscope  the  object 
to  be  looked  at  is  placed  within  the  proper  focal  distance  from  the 
objective  and  a  real  magnified  image  is  then  formed  in  the  interior 
of  the  draw-tube.  The  eye  sees  this  through  the  ocular  or  eye- 
piece, which  again  enlarges  the  real  image  in  the  draw-tube,  and 
forms  in  the  eye  a  highly  magnified  visual  image.  This  is  the  optical 
principle  of  the  construction  and  use  of  the  microscope. 


90  METHODS  OF  OBSERVING  BACTERIA 

Lenses. —  The  objectives  commonly  used  in  work  in  histology, 
pathology,  and  bacteriology  generally  have  focal  distances  of  two- 
thirds  inch,  one-sixth  inch,  and  one-twelfth  inch  (16  mm.,  4  mm., 
2  mm.),  and  they  magnify,  if  used  with  the  proper  eyepieces,  about 
80,  400,  and  800  times  or  linear  diameters. 

Dry  Lenses. — The  first  two  of  these  three  lenses  are  used  without 
the  interposition  of  any  fluid  between  the  cover-glass  of  the  prep- 
aration and  the  front  lens  of  the  objective,  and  are  called  dry  lenses. 

Immersion  Lenses. — The  third  one,  that  which  has  the  highest 
magnification  and  the  shortest  focal  distance,  is  used  with  a  drop 
of  thickened  cedar  oil,  called  homogeneous  immersion  oil,  interposed 
between  the  cover-glass  of  the  preparation  and  the  front  lens  of  the 
objective.  Lenses  used  in  this  manner  are  called  homogeneous  immer- 
sion lenses.  High-power  lenses  are  best  constructed  as  immersion 
lenses  for  optical  reasons.  When  a  preparation  is  examined  the  light 
is  first  reflected  upward  by  the  mirror  of  the  substage  illuminating 
apparatus,  it  then  passes  the  opening  in  the  iris  diaphragm,  enters 
the  condenser  proper,  and  is  focussed  by  it  upon  the  microscopic 
object  to  be  examined.  From  the  latter  the  rays  of  light  enter  the 
objective  of  the  microscope.  In  doing  so,  when  a  dry  lens  is  used, 
the  rays  of  light  on  leaving  the  cover-glass  of  the  microscopic  prep- 
aration, pass  through  a  small  amount  of  air  before  reaching  the 
lower  lens  of  the  objective,  and  are  refracted  in  such  a  manner  that  a 
considerable  portion  of  the  light  is  lost.  This  loss  of  light  and  the 
decrease  in  the  angle  of  aperture  under  which  the  rays  enter  the 
objective  cause  loss  of  clearness  and  detail  in  the  picture.  If,  instead 
of  air  between  the  cover-glass  and  the  front  lens  of  the  objective 
there  is  a  drop  of  thickened  cedar  oil,  which  acts  toward  light  in 
the  same  manner  as  the  cover-glass  of  the  microscopic  preparation, 
or,  in  other  words,  possesses  the  same  refractive  index  as  ordinary 
glass,  there  will  be  no  loss  of  light,  but  a  larger  angle  of  aperture  of 
the  entering  rays  and  a  much  better  image.  Therefore,  high-power 
lenses  are  constructed  as  homogeneous  oil-immersion  lenses,  and  such 
a  lens  with  a  numerical  aperture  angle  of  1.4  is  better  than  one 
with  a  numerical  aperture  of  1.3.  Homogeneous  immersion  lenses 
have  a  very  short  focal  distance  (2  mm.  =  TT¥  inch);  hence,  if  a 
thick  cover-glass  be  used  it  will  be  impossible  to  bring  the  lens  within 
the  proper  focal  distance  of  the  objects  (bacteria)  to  be  looked  at. 
It  is,  therefore,  necessary  to  use,  in  work  with  bacteria,  the  thinnest 
cover-glasses,  that  is,  No.  1.  No.  2  may  sometimes  answer,  but  it 
is  not  safe  to  use  them,  and  the  student  should  always  see  that  he 
uses  for  work  with  bacteria  the  thinnest  kind. 

Focussing.— In  order  to  see  clearly  with  the  microscope,  it  is  always 
necessary  to  lower  or  raise  the  tube  in  such  a  manner  that  the  ob- 
ject has  the  proper  focal  distance  from  the  front  lens  of  the  objective. 
The  proper  adjustment  must  be  judged  by  the  eye.  This  manipu- 
lation of  the  instrument  to  obtain  a  clear  picture  is  called  focussing, 


STAINED  AND  UNSTAINED  OBJECTS  91 

that  is,  getting  the  preparation  or  object  into  focus.  The  three  ob- 
jectives of  a  good  modern  microscope  which  are  attached  to  the 
triple  nosepiece  or  revolver  are  parfocal  and  correctly  and  identi- 
cally centred.  The  term  parfocal  may  be  best  explained  as  follows: 
Suppose  an  object  is  first  focussed  with  the  f-inch  (16  mm.)  or  low- 
power  lens.  The  nosepiece  is  now  swung  around  so  that  the 
J-inch  (4  mm.)  higher  power  lens  replaces  the  J-inch  lens  under  the 
tube.  If  the  two  lenses  are  arranged  on  the  revolver  in  an  absolutely 
parfocal  manner  the  ^-inch  lens,  after  the  turn  is  made,  should  be 
strictly  in  focus.  This,  however,  is  rarely  the  case,  and  it  is  necessary 
to  use  the  fine  adjustment  to  get  a  really  sharp  focus.  So  the  term 
parfocal  has  a  relative  value  and  meaning  only.  The  term  perfectly 
and  identically  centred  means  that  the  centre  of  all  objectives  is  in 
the  optical  axis  of  the  instrument,  so  that  an  object  which  is  exactly 
in  the  centre  of  one  objective  will  also  be  exactly  in  the  centre  of 
another  objective  if  the  latter  is  swung  around  to  the  place  of  the 
former  by  turning  the  revolver  or  nosepiece.  Here,  likewise,  instead 
of  absolute  correctness  only  a  more  or  less  close  approximation  is 
attained.  Working  with  bacteria  a  f-inch  and  a  y^-inch  objective 
are  generally  used.  The  ^-inch  objective,  except  for  the  observation 
of  the  ray  fungus  in  pus,  is  generally  employed  in  section  work  in 
histology  or  pathology. 

In  studying  bacterial  preparations  the  |- inch  dry  lens  is  first  used 
for  a  general  orientation  of  the  specimen  and  to  pick  out  a  spot  which 
is  neither  too  much  overloaded  with  bacteria  nor  overstrained.  The 
supply  of  organisms,  however,  should  not  be  scanty  nor  understained. 
The  oil-immersion  lens  is  now  brought  into  use.  It  is  not  advisable 
to  swing  around  the  T^-inch  lens  and  depend  upon  its  being  par- 
focal  with  the  f-inch  lens.  Often  it  is  not,  and  by  swinging  it  around 
after  the  low-power  objective  has  been  in  focus  it  may  strike  against 
the  cover-glass  or  some  overhanging  margin  of  the  squeezed-out 
Canada  balsam.  The  glass  may  damage  the  expensive  high-power 
lens,  and  the  balsam  will  certainly  soil  it  and  necessitate  its  being 
cleaned.  It  is  best  to  raise  the  tube  before  the  oil-immersion  lens  is 
swung  into  place.  Good  modern  microscopes  for  work  in  bacteri- 
ology and  hematology  are  often  supplied  with  an  attachable  mech- 
anical stage,  which  permits  of  a  systematic  search  over  the  whole  of 
the  microscopic  preparation.  However,  instruments  for  the  use  of 
students  in  their  laboratory  training  are  rarely  supplied  with  this 
desirable  accessory.  There  are  also  some  devices  called  warm  or 
heated  stages  which  permit  the  study  of  bacteria  and  other  micro- 
organisms at  higher  stationary  temperatures. 

Stained  and  Unstained  Objects. — An  important  point  in  microscopic 
work  which  the  student  should  always  recollect  is  the  following: 
When  stained  objects  are  examined  the  colored  image  shows  better 
the  more  powerful  the  illumination.  Hence,  in  the  examination  of 
stained  preparations  the  iris  diaphragm  must  be  kept  wide  open. 


92 


METHODS  OF  OBSERVING  BACTERIA 


On  the  other  hand,  with  unstained  bacteria,  cells  and  other  small  bodies 
in  general,  the  details  of  the  pictures  depend  upon  the  difference  in 


FIG.  35 

EYE-PIECE  OR  OCULAR. 


DRAW-TUBE  WITH  MM.  SCALE. 


COARSE  ADJU8TM. 


FINE  ADJUSTM. 

OR 
MM.   SCREW. 


A  modern  student's  and  practitioner's  microscope. 

refraction   between   the   different   parts   of    the    bacteria   (flagellse, 
granules,  spores,  capsules,  etc.)  and  the  medium  in  which  they  are 


MICROSCOPE  FOR  STAINED  BACTERIAL  PREPARATIONS     93 

suspended  (water,  bouillon,  milk,  wine,  pus,  etc.).  These  slight 
differences  would  be  lost  in  a  flood  of  light  and  the  iris  diaphragm 
should  be  closed  considerably.  It  is  much  easier  for  the  beginner 
to  examine  stained  preparations  of  bacteria  than  the  unstained  ones, 
hence  the  steps  in  the  former  procedure  will  be  given  first. 

Steps  in  Using  the  Microscope  for  Stained  Bacterial  Preparations. — 
1.  Place  the  instrument  in  front  of  the  source  of  light  in  a  vertical 
(not  inclined)  position  and  deposit  the  preparation  on  the  stage  of 
the  microscope.  See  that  the  -surfaces  of  the  objectives  and  eye- 
pieces are  free  from  dust  and  grease  and  otherwise  clean. 

2.  Bring  the  f-inch  low-power  lens  into  the  centre  and  lower 
the  tube  on  the  coarse  adjustment  so  that  it  is  about  one  inch  from 
the  cover-glass. 

3.  Illuminate  the   object   by  manipulating  the  plane  reflector  (do 
not  use  the  concave  mirror).     Have  iris  diaphragm  wide  open  and 
slowly  lower  the  tube  on  the  coarse  adjustment  until  the  color  of  the 
object  can  be  well  recognized.     Now,  again  manipulate  the  reflector 
and  lower  the  Abbe  condenser  until  the  field  of  vision  is  well  and 
uniformly  illuminated  and  until  no  shadows  of  the  window  frame 
or  parts  of  the  lamp  are  seen  in  the  field  of  vision. 

4.  Move  the  microscopic  slide    around    until    a  place  is   found 
where  the  stain  is  neither  too  dense  nor  too  scanty.    Place  this  spot 
in  the  centre  of  the  field  and  clamp  down  the  slide  so  that  it  is  immov- 
able.   Look  again  to  see  whether  the  selected  spot  is  still  exactly  in 
the  centre  of  the  field;  if  not,  move  the  slide  under  the  clamps  until 
the  desired  spot  is  where  it  is  wanted. 

5.  Raise  the  tube  somewhat  on  the  coarse  adjustment  and  swing 
the  y^inch  oil-immersion  lens  into  the  centre.    Place  a  drop  of  cedar 
immersion  oil  on  the  centre  of  the  cover-glass;  run  the  immersion 
lens  on  the  coarse  adjustment  into  the  oil.     Raise  the  tube  on  the 
coarse  adjustment  so  that  the  drop  of  cedar  oil  is  drawn  out.    The 
oil-immersion  lens   is  now  considerably  above  the  proper  focal  dis- 
tance of  the  object. 

6.  Raise  the  substage  condenser  as  high  as  it  will  go.     (Its  front 
lens  is  now  on  a  level  with  the  stage  of  the  microscope.)    Again,  man- 
ipulate the  plane  mirror  so  that  the  field  is  well  and  uniformly  lighted. 
Now,  slowly,  with  the  coarse  adjustment,  lower  the  tube  under  the 
guidance  of  the  eye  which  watches  the  field  of  vision  through  the 
ocular.      As  soon  as  some  color  is  observed  the  fine  adjustment  or 
micrometer  screw  must  be  used  until  a  clear  image  is  obtained;  that 
is,  until  the  oil-immersion  lens  is  sharply  in  focus. 

The  microscopic  picture  so  obtained  may  sometimes  still  be  im- 
proved by  lowering  the  substage  condenser  a  very  little  bit,  and  by 
closing  the  iris  diaphragm  somewhat;  but  the  beginner  should  be 
careful  in  attempting  these  corrections,  which  require  a  good  deal 
of  skill  and  judgment  in  the  interpretation  of  the  image. 

The  danger  for  the  beginner  in  the  use  of  the  oil-immersion  lens 


94          METHODS  OF  OBSERVING  BACTERIA 

consists  in  lowering  it  so  much  that  it  is  brought  beyond  the  proper 
focal  distance.  He  is  then  likely  to  lower  it  still  farther  until  the 
front  lens  touches  the  cover-glass  and  the  pressure  may  be  great 
enough  to  crack  or  dislocate  the  former.  The  expensive  objective  is 
then  ruined  and  can  only  be  repaired  at  a  considerable  outlay  of 
money. 

FIG.  36 


Hollow  slide  with  cover-glass. 

Steps  in  Using  the  Microscope  for  Unstained  Bacteria  in  the  Hanging 
Drop  or  Moist  Chamber.1 — 1.  The  same  as  step  No.  1  for  stained 
preparations. 

2.  The  same  as  step  No.  2  for  stained  preparations. 

3.  Illuminate  the  object — that  is,  the  live  bacteria  in  the  hanging 
drop  of  water  or  bouillon — by  manipulating  the  plane  reflector  (do 
not  use  the  concave  mirror).     Have  the  iris  diaphragm  closed  sb  that 
the  field  is  only  very  dimly  lighted.     Slowly  lower  the  tube  on  the 
coarse  adjustment  until  the  margin  of  the  drop  can  be  recognized. 
Move  the  margin  of  the  drop  near  the  centre  of  the  field  so  that  the 
centre  of  the  lens  is  just  over  the  thinnest  portion  of  the  drop.    Clamp 
the  slide  and  see  once  more  whether  the  desired  spot  is  in  the  centre 
of  the  field.     (All  this  has  to  be  done  under  guidance  of  the  eye, 
looking  through  the  low-power  dry  lens.) 

4.  Raise  the  substage  condenser  to  its  highest  level;  raise  the  tube; 
place  a  drop  of  immersion  oil  on   the  cover-glass,  which  is  situ- 
ated over  the  concavity  of  the  slide.     Now  bring  the  oil-immersion 
lens  into  place  and  lower  it  until  it  touches  the  cedar  oil.    Keep  on 
lowering  the  lens  on  the  coarse  adjustment,  very  carefully  and  slowly, 
until  it  firmly  touches  the  cover-glass.     This  can  be  ascertained  by 
watching  the  vaselin  which  is  between  the  cover-glass  and  the  slide. 
As  soon  as  the  oil-immersion  objective  presses  firmly  on  the  coyer- 
glass  resting  over  the  concavity  the  vaselin  will   be   squeezed  out 
from  under  it.    In  so  manipulating  the  immersion  lens  there  is  prac- 
tically no  danger  of  injuring  it,  because  a  little  excess  pressure  will 
crack  the  cover-glass,  due  to  the  poor  backing  given  it  by  the  con- 
cavity of  the  slide.     If  this  occurs  the  hanging  drop  will  have  to  be 
made  over  again. 

5.  After  the  immersion  lens  has  been  lowered  as  described  it  is 
below  the  proper  focal  distance.    Open  the  iris  diaphragm  and  see 
that  the  field  is  well  and  uniformly  lighted  (if  this  is  the  case  there 
is  no  danger  of  having  an  oblique  illumination).    Now,  close  the  iris 
diaphragm  so  that  only  a  small  opening  is  left  and  the  field  is  very 

1  For  steps  in  preparing  moist  chamber,  see  p.  103. 


MICROMETERS 


95 


dimly  lighted.  Raise  the  tube  on  the  coarse  adjustment  very  slowly 
and  gradually.  As  soon  as  the  faintest  details  can  be  seen,  perhaps 
the  margin  of  the  drop  or  a  few  individual  bacteria,  use  the  fine 
adjustment  to  get  the  exact  focus. 

In  examining  both  stained  and  unstained  bacterial  preparations 
it  is  desirable  to  move  the  slide  in  order  to  examine  the  whole  speci- 
men; this  necessitates  constant  changing  of  the  fine  adjustment  in 
order  to  keep  the  object  in  focus. 


FIG.  37 


Attachable  mechanical  stage. 

Micrometers. — In  the  study  of  bacteria  their  size  is  always  men- 
tioned. The  task  of  measuring  so  small  an  object  as  a  bacterium 
must  appear  a  very  formidable  affair  to  the  student,  yet  it  is  an 
exceedingly  simple  procedure.  It,  however,  requires  the  use  of 
some  microscopic  accessories,  such  as  a  stage  micrometer  and  an 
eyepiece  micrometer.  The  stage  micrometer  consists  of  a  glass  slide 
with  a  finely  ruled  scale  of  one  millimeter,  divided  into  100  equal 
parts,  hence  the  space  between  two  dividing  lines  is  equal  to  one- 
hundredth  of  a  millimeter,  or  10/z  =  10  micra  =  10  micromillimeters. 
The  simplest  eyepiece  micrometer  consists  of  a  circular  glass  disk 
with  a  finely  ruled  scale  which  divides  a  line  in  the  centre  of  the  disk 
into  fifty  equal  portions.  This  disk  should  be  laid  upon  the  inner 
diaphragm  of  the  eyepiece.  In  order  to  do  this  the  upper  lens  of 
the  eyepiece  must  be  unscrewed  and  then  replaced  again.  Some 
eyepiece  micrometers  are  very  complicated  and  expensive,  but  all 
are  made  on  the  same  principle,  a  line  in  the  centre  divided  into 


96  METHODS  OF  OBSERVING  BACTERIA 

equal  spaces.  The  steps  in  the  use  of  the  two  micrometers  for  the 
purpose  of  measuring  bacteria,  cells,  and  other  very  small  micro- 
scopic objects  are  as  follows: 

1.  Unscrew  the  upper  lens  of   the  eyepiece,  which  exposes    the 
diaphragm  of  the  latter.     Place  the  circular  glass  disk  micrometer, 
generally  held  in  a  small  metallic  frame,  with  its  flat  side  downward 
upon  the  diaphragm,  replace  the  front  lens  of  the  eyepiece  and  return 
to  its  proper  position  on  the  draw  tube  of  the  microscope.    Regulate 
the  upper  lens  of  the   eyepiece  so  that  the  lines  of  the  eyepiece 
micrometer  show  clearly. 

2.  Place  the  stage  micrometer  on  the  stage  and  get  its  scale  in 
focus  with  the  oil-immersion  objective.    This  must  be  done  according 
to  the  rules  for  examining  unstained  objects,  hence  the  .iris  diaphragm 
must  be  closed  as  much  as  possible. 

3.  The  scale  of  the  stage  micrometer  runs  from  above  downward 
in  the  field  of  vision.      Now  manipulate  the  eyepiece  micrometer, 
by  rotating  the  eyepiece,  so  that  the  ruled  scale  of  the  latter  also 
runs  from  above  downward.    Next,  move  the  stage  micrometer  (this 
can  be  more  easily  done  if  the  microscope  has  an  attachable  mech- 
anical stage)  so  that  the  first  line  of  its  scale  and  the  first  line  of  the 
eyepiece  micrometer  fall  together  (overlap  each  other). 

4.  Manipulate  the  draw-tube  so  that  a  number  of  subdivisions 
of  the  eyepiece  micrometer  are  just  equal  to  one  partition — that  is, 
the  space  between  two  lines — of  the  stage  micrometer. 

5.  Suppose  that  eight  of  the  spaces  of  the  eyepiece  micrometer 
are  equal  to  one  space  of  the  stage  micrometer;  then  each  space  of 
the  eyepiece  micrometer  with  the  magnification   used  is  equal  to 
1.25  micra.    As  already  stated,  the  stage  micrometer  is  so  ruled  that 
1  mm.  is  divided  into  100  equal  parts;  therefore,  each  space  is  equal 
to  10  micra,  and  8  eyepiece  spaces  =10  micra;  hence,  1  space=  1.25 
micra. 

6.  Raise  the  tube  of  the  microscope  on  the  coarse  adjustment; 
remove  the  stage  micrometer  and  replace  it  by  the  stained  cover- 
glass  preparation  of  bacteria  which  are  to  be  measured.     Open  the 
iris  diaphragm  and  bring  the  bacteria  into  focus. 

7.  Now  manipulate  the  slide  on  the   stage  and  the  eyepiece  by 
rotating  it  so  that  a  typical  bacterium,  say  a  bacillus,  just  stands  at 
right  angles  to  the  lines  of  the  ruling  of   the  eyepiece  micrometer. 
See  that  the  end  of  the  bacterium  apparently  just  touches  one  of  the 
lines  and  note  over  how  many  divisions  the  bacillus  extends. 

8.  Suppose  that  the  bacillus  just  fills  three  divisions  of  the  scale; 
then  it  is  three  times  1.25  micron  or  3.75  micra  long. 

9.  In  making  these  measurements   the  following  precautions  are 
to  be  used.     The  slide  on  which  the  bacterial  preparation  is  made 
should  be  the  same  thickness  as  the  stage  micrometer  slide.     After 
the  draw-tube  has  been  adjusted  in  order  to  bring  a  whole  number 
of  divisions  of  the  eyepiece  into  one  division  of  the  scale  of  the  stage 


DARK-FIELD  ILLUMINATOR  97 

micrometer,  the  draw-tube  must  be  left  exactly  where  it  is.  If  it 
had  been  drawn  out  say  to  171.5  millimeters,  as  shown  by  its  scale, 
it  must  be  left  there  after  the  removal  of  the  stage  micrometer  and 
the  substitution  of  the  slide  with  the  bacteria.  A  large  number  of 
the  most  typical  forms  (not  involution  forms)  must  be  measured  and 
the  size  given  in  terms  of  the  minimum  and  maximum  length,  for 
instance,  from  2.5  to  3.75  micra. 

Camera  Lucida. — The  camera  lucida  drawing  apparatus  and  the 
photomicrographic  camera  are  important  microscopic  accessories  in 
the  study  of  bacteria.  The  use  of  the  former  is  very  simple  and 
explains  itself;  that  of  the  latter,  however,  cannot  here  be  taken  up 
in  detail,  since  it  requires  special  studies  and  much  delicate  work  in 
order  to  obtain  good  photomicrographs.  When  they  are  prepared 
with  skill  they  are  by  far  the  best  method  of  illustrating  micro- 
organisms. 


Camera  lucida  drawing  apparatus. 

Dark-field  Illuminator. — A  microscopic  accessory  which  has  recently 
come  very  prominently  into  use  is  the  dark-field  illuminator,  devised 
for  observing  living  bacteria  and  other  microscopic  objects.  It 
shows  these  as  the  only  light  objects  in  an  otherwise  perfectly  dark 
field  of  vision.  When  it  is  to  be  used  the  condenser  lens  of  the  sub- 
stage  illuminating  apparatus  of  the  microscope  must  be  removed  and 
the  dark-field  illuminator  placed  either  on  top  of  the  stage  or  slipped 
into  the  position  that  the  Abbe  condenser  lens  previously  occupied. 
Instruments  are  designed  to  work  in  either  one  position  or  the  other, 
but  when  constructed  for  use  on  top  of  the  stage  they  cannot  be  used 
below  it,  and  vice  versa.  The  dark-field  illuminator  is  so  arranged 
that  it  will  refract  the  parallel  rays  of  light  which  are  reflected  upward 
by  the  mirror,  so  that  they  will  be  changed  into  very  oblique  rays. 
These,  when  they  strike  the  cover-glass  of  the  preparation,  suffer  what 
is  known  as  total  reflection;  that  is,  they  are  refracted  back  in  the 
direction  from  which  they  came;  in  consequence  of  this  the  field  of 
7 


98 


METHODS  OF  OBSERVING  BACTERIA 


the  microscope  appears  perfectly  dark.     However,  the  very  central 
rays  of  light  which  strike  the  cover-glass  illuminate  the  bacteria  and 


FIG. 


Dark-field  illuminator,  or  reflecting  condenser. 
FIG.  40 


if    I    if 

ii  i  1 

Optical  construction  of  dark-field  illuminator. 


FIG.  41 


Electric  arc  lamp  with  hand  feed  for  a  current  of  4  amperes  and  illuminating  lens  to  be  used 
with  the  dark-field  illuminator. 


DARK-FIELD  ILLUMINATOR  99 

small  particles  and  these  then  stand  out  as  very  bright  objects  on  an 
entirely  dark  background.  As  a  source  of  light  for  the  dark-field 
illuminator  we  can  use  an  inverted  Welsbach  light,  immediately 
before  the  reflecting  mirror  of  the  microscope;  or  still  better,  a  small 
electric-arc  light  especially  constructed  for  the  purpose.  Whichever 
light  is  used,  its  rays  must  be  collected  by  a  condensing  lens,  so  that 
the  mirror  receives  a  very  strong  bundle  or  pencil  of  rays  of  light. 
The  use  of  the  dark-field  illuminator  is  as  follows: 


FIG.  42 


Dark-field  illuminator  used  with  Welsbach  gas  light. 

1.  Arrange  the  dark-field   illuminator   according  to  the    type    of 
apparatus,  either  on  top  of  the  stage  or  below  it. 

2.  Place  a  drop  of  the  fluid  to  be  examined  on  a  clean  slide  and 
cover  with  a  clean  No.  1  cover-glass. 

3.  Place  a  drop  of  immersion  oil  in  the  centre  of  the  upper  surface 
of  the  dark-field  illuminator. 

4.  Place  slide  on  upper  surface  of  dark-field  illuminator.    There 
is  now  no  air  between  the  latter  and  the  slide,  since  these  surfaces 
have  the  immersion  oil  between  them. 

5.  Place  the  instrument  so  that  the  light  of  the  inverted  Welsbach 
burner  or  electric  arc  light  is  near  the  refracting  mirror  of  the  substage 
of  the  microscope,  and  place  between  the  source  of  light  and  the 
reflector  the  concave  lens  which  collects  the  rays  of  light.     When 


100 


METHODS  OF  OBSERVING  BACTERIA 


an  electric-arc  lamp  is  used  the  condensing  lens  is  combined  with  it; 
when  a  Welsbach  burner  is  used  the  lens  is  separate. 

6.  Manipulate  the  reflector  so  that  the  microscopic  field  appears 
uniformly  dark,  while  cells,  bacteria,  granules,  etc.,  appear  as  exceed- 
ingly light,  highly  refractive  objects. 

The  dark-field  illuminator  is  not  at  all  difficult  to  use,  and  with  it 
fine  objects,  such  as  spirochetse,  etc.,  are  found  much  more  easily 
than  with  the  ordinary  hanging-drop  or  moist-chamber  method. 
The  high-power  dry  lenses  ^  and  ^-inch  focal  distance  are  gen- 
erally best  for  use  with  the  dark-field  illuminator.  There  is^no  great 
advantage  in  the  use  of  oil-immersion  lenses.  If  they  are  to  be  used 
at  all  it  is  necessary  to  screw  into  the  interior  of  the  lens  a  small 
metal  funnel  in  order  to  limit  the  field  of  vision;  this  is  necessary  for 
optical  reasons.  It  is  also  necessary  to  place  a  drop  of  immersion  oil 
on  the  upper  surface  of  the  cover-glass  in  addition  to  the  drop  which 
was  placed  on  the  upper  surface  of  the  dark-field  condenser.  The 
latter  is  generally  supplied  with  a  diaphragm  to  regulate  the  amount 
of  light  admitted. 


FIG.  43 


Glass  slide. 


FIG.  44 


FIG.  45 


Square  and  round  cover-glass. 

Small  Utensils. — A  number  of  small  appliances  and  utensils  are 
necessary  for  the  first  elementary  studies  in  bacteriology. 

Slides  and  Cover-glasses. — Slides  and  cover-glasses  are  used 
similarly  as  in  normal  and  pathologic  histology.  It  has  previously 
been  pointed  out  that  the  cover-glasses  or  cover-slips  for  use  in  work 
with  bacteria  should  be  the  thinnest  kind,  that  is,  No.  1  (they  are 
from  0.15  to  0.17  mm.  in  thickness).  Both  slides  and  covers  should 
be  very  clean  and  free  from  dirt  and  grease.  As  they  are  generally 


SMALL  UTENSILS 


101 


furnished  they  are  free  from  neither.     The  best  method  to  cleanse 
them  which  will  also  remove  any  soluble  alkalies  adhering  to  the 


FIG.  46 


Stewart's  cover-glass  forceps. 
FIG.  47 


FIG.  48 


Cornet's  cover-glass  forceps. 

glass,  fresh  from  the  factory,  is  the  following:  Immerse  cover- 
glasses  and  slides  first  in  water  acidulated  with  a  mineral  acid  (HC1, 
HNO3,  H2SO4) ;  wash  well  in  ordinary  tap 
water  to  remove  every  trace  of  acid,  then 
wash  in  alcohol,  and  wipe  dry  with  a 
soft  clean  rag  or  with  Japanese  tissue 
paper. 

Forceps. — In  order  to  prepare  properly 
a  stained  cover-glass  specimen  it  is  neces- 
sary to  hold  it  in  a  pair  of  small  forceps. 
Those  used  most  frequently  for  work  of 
this  kind  are  the  Cornet  or  Stewart  forceps 
or  some  of  their  modifications. 

Platinum  Rod. — The  most  important  in- 
strument of  the  bacteriologist  is  the  plati- 
num rod.  It  consists  of  a  slender  glass 
rod  about  eight  to  ten  inches  long,  into 
one  end  of  which  has  been  fused  a  piece 
of  platinum  wire  about  twelve  to  fourteen 
inches  long.  For  ordinary  work  the  plati- 
num wire  is  rather  thin  (No.  26  or  No.  27); 
for  special  work  it  is  well  to  have*  also  a 
strong  platinum  wire  which  can^j  easily 
perforate  a  tissue.  The  wire  of  the  plati- 
num rod  is  either  used  straight  as  a  needle:  ^  Platinum  needle  cand  .IO°P- 

..,  ,  ,  •       *  i        mi          For  most   purposes   finer  wire  is 

or  with  a  round  loop  on  its  free  end.    The    used, 
latter  arrangement   is   termed   the  plati- 
num loop  and  is  generally  used  in  cover-glass  preparations  from 
pus,  other  fluids,  or  pure  cultures. 


102 


METHODS  OF  OBSERVING  BACTERIA 


Lamps. — The  next  utensil  required  is  a  small  alcohol  lamp  or 
Bunsen  gas  burner.  One  or  the  other  is  necessary  for  sterilizing 
the-platinum  rod,  which  is  never  used  unless  it  has  previously  been 
heated  in  a  flame  and  never  put  aside  until  it  has  gone  through 
the  same  process.  This  is  the  only  way  to  avoid  contamination  of 
preparations  made  with  -outside  microorganisms  with  which  the 
platinum  loop  has  come  in  contact  while  in  use. 


FIG.  49 


FIG.  50 


Bunsen  burners. 

Glassware. — The  dyes  used  in  staining  cover-glass  preparations 
are  best  kept  in  small  bottles  provided  with  droppers.  Aside  from 
these,  small  dishes  are  needed  in  which  the  cover-glass  preparations 
can  be  washed  in  water  or  alcohol.  Small  beakers  or  whiskey 
glasses  will  do  for  this  purpose.  To  complete  the  outfit  for  elementary 
work,  some  small  funnels,  small  round  filters,  and  filter  paper  are 
necessary,  as  it  is  sometimes  desirable  to  filter  the  stain  directly 
before  use  on  the  cover-glass.  A  Canada-balsam  bottle  and  somfc 


FIG.  51 


FIG.  52 


Canada-balsam  bottle. 


Three  staining  solution  bottles  with  droppers. 


so-called  concave  slides  are  also  needed.  The  latter  are  slides 
generally  a  little  thicker  than  ordinary  slides,  with  one  or  two 
concavities  ground  in  the  glass,  and  used  in  making  hanging-drop 
preparations  in  studying  live  bacteria. 

Method  of  Preparing  a  Hanging  Drop  or  Moist  Chamber  or  Concave 
Slide. — The  following  are  the  steps  in  preparing  a  hanging  drop  for 
studying  bacteria  and  other  microorganisms  unstained  and  in  the 
live  state: 


QUESTIONS  103 

1.  Paint  with   a  earners-hair  brush,  around  the  concavity  of   a 
concave  slide,  a  ring  of  vaselin. 

2.  Clean  cover-glass    particularly  well,   so   that   it   is   free   from 
grease;  hold  in  a  pair  of  forceps,  and  with  a  platinum  loop,  place  on 
the  centre  of  the  glass  a  small   drop  of  water,  or,  better,  sterile 
physiologic  salt  solution. 

3.  Enter  culture   tube  with   sterile  platinum  loop   and    remove 
from  the  surface  of  the  agar,  gelatin,  etc.,  a  small  bit  of  the  growth, 
avoiding  at  the  same  time  to  take  any  of  the  culture  medium. 

4.  Rub  up  the  small   bit  of  growth  with  the  drop  of  water  on 
the  cover-glass,  so  that  there  is  formed  a  uniform  emulsion  of  the 
bacteria  in  water.     Spread  the  drop  out  considerably  so  that  it  is  as 
shallow  as  possible. 

5.  As  soon  as  this  is  accomplished,  place  the  cover-glass  over  the 
concavity  of  the  concave  slide  in  such  manner  that  the  drop  hangs 
down  free  into  the  hollow  space. 

6.  Now  press  cover-glass  down  gently  into  the  vaselin  surround- 
ing the  concavity,  so  that  the  latter  is  closed  air-tight. 

7.  The  hanging  drop  is  now  ready  to  be  examined  in  the  manner 
described  above. 

When  bacteria  in  a  fluid  excretion  like  urine,  or  from  a  fluid  culture 
medium  like  nutrient  bouillon,  are  to  be  examined  a  drop  of  these 
fluids  can  be  placed  directly  upon  the  cover-glass  without  first  apply- 
ing a  drop  of  water. 

QUESTIONS. 

1.  What  is  meant  by  a  pure  culture  of  a  bacterium? 

2.  What  is  a  colony  of  a  pure  culture? 

3.  At  what  time  in  the  development  of  a  bacterial  growth  can  colonies  best 
be  studied? 

4.  What  properties  of  bacteria  in  pure  cultures  can  be  recognized  without 
the  aid  of  the  microscope? 

5.  What  sources  of  light  are  employed  in  the  use  of  the  microscope  ? 

6.  What  are  the  main  parts  of  a  modern  compound  microscope  adapted  for 
use  in  work  with  bacteria  ? 

7.  Explain   the    terms:    ocular,    objective,   revolver,   npsepiece,   draw-tube, 
coarse  adjustment,  micrometer  screw,  Abbe  condenser,   iris  diaphragm,   plane 
mirror,  concave  reflector,  mechanical  stage,  oil-immersion  lens,  dry  lens,  focal 
distance. 

8.  For  what  distance  are  the  objectives  corrected? 

9.  How  far  should  the  draw  tube  be  drawn  out  and  why? 

10.  What  is  meant  by  spherical  aberration  of  an  objective?    What  by  chrom- 
atic aberration? 

11.  What  is  meant  by  the  refraction  of  light? 

12.  How  is  it  affected  by  transparent  media  of  various  densities? 

13.  What  is  meant  by  the  focus  of  an  objective  or  its  focal  distance? 

14.  In  what  relation  does  the  focal  distance  stand  to  the  magnification? 

15.  Why  is  a  high-power  homogeneous  immersion  lens  better  than  a  dry  lens 
of  the  same  focal  distance  ? 

16.  Explain  why  very  thin  cover-glasses  are  used  with  the  TV  oil-immersion 
objective? 

17.  How  is  the  iris  diaphragm  used  when  examining  stained   and  unstained 
bacteria,  and  why? 

18.  What  does  the  term  parfocal  mean? 


104         METHODS  OF  OBSERVING  BACTERIA 

19.  What  is  meant  by  exactly  and  evenly  centred  objectives? 

20.  Give  in  detail  the  steps  in  the  examination  of  a  stained  bacterial  cover- 
glass  preparation  with  the  microscope. 

21.  Give  the  steps  in  using  the  microscope  when  examining  a  hanging-drop 
preparation.  « 

22.  How  can  the  size  of  bacteria  be  measured? 

23.  What  is  (a)  stage  micrometer?    (6)  eyepiece  or  ocular  micrometer? 

24.  Describe  the  use  of  these  microscopic  accessories  in  measuring  bacteria. 

25.  What  is  a  camera  lucida?    What  is  a  photomicrographic  camera? 

26.  What  is  a  dark-field  illuminator? 

27.  How  does  the  microscopic  image  look  when  a  dark  field  illuminator  is 
used? 

28.  Describe  the  use  of  the  dark -field  illuminator. 

29.  What  is  a  platinum  rod? 

30.  What  is  a  concave  slide? 

31.  What  is  a  Stewart  forceps? 

32.  Describe  the  method  of  cleaning  slides  and  cover  glasses. 

33.  Describe  the  method  of  preparing  a  hanging  drop  in  a  concave  slide. 


CHAPTER    IX. 

STAINING  OF  BACTERIA  IN  COVER-GLASS  PREPARATIONS  AND 

IN  TISSUES. 

Anilin  Stains. — In  laboratory  work  in  histology  and  pathology 
eosin  is  used  as  a  so-called  counter-stain;  this  dye  is  one  of  the  anilin 
stains.  These  stains  are  complicated  bodies  derived  from  anilin  oil, 
a  heavy  liquid  which  is,  however,  not  a  true  oil,  but,  according  to 
its  chemical  properties,  an  alkali  or  a  base,  like  caustic  soda,  caustic 
potash,  or  ammonia.  In  fact,  it  contains  the  ammonia  radical  in  its 
molecule.  By  combining  the  basic  anilin  oil  with  various  acids  in 
certain  proportions  either  a  neutral,  an  acid,  or  an  alkaline  or  basic 
salt  may  be  obtained.  Eosin,  mentioned  above,  is  an  acid  anilin 
stain.  Bacteria,  however,  are  particularly  well  stained  by  basic  or 
alkaline  anilin  stains.  The  most  useful  of  these  for  work  of  this 
kind  are  gentian  violet,  fuchsin,  and  methylene  blue.  It  is  best  to  keep 
them  on  hand  in  the  laboratory  in  the  form  of  filtered  saturated 
alcoholic  solutions,  since  these  do  not  decompose  or  deteriorate. 
Saturated  alcoholic  solutions  contain  about  25  grams  of  the  dry  stain 
to  100  c.c.  of  alcohol.  Watery  solutions  are  prepared  for  use  from 
the  alcoholic  stock  solutions  by  adding  to  100  c.c.  of  distilled  water 
about  5  to  10  c.c.  of  the  saturated  alcoholic  solution.  It  should  be 
remembered  that  a  gentian  violet  in  watery  solution  stains  very  rapidly, 
and  easily  overstains.  Methylene  blue  stains  rather  slowly,  and  there- 
fore does  not  easily  overstain.  Fuchsin  takes  an  intermediate  position. 
Hence,  the  time  for  staining  is  as  follows : 

With  watery  gentian  violet  solution 1J^  to  21A  minutes 

With  watery  fuchsin  solution 3      to  4      minutes 

With  watery  methylene-blue  solution 5      minutes  or  longer 

Staining  Bacteria  on  Cover-glass  Preparations. — In  staining  bacteria 
with  the  simple  watery  anilin  stains,  say  in  pus  or  in  any  other  dis- 
charge or  excretion,  like  urine,  feces,  etc.,  proceed  as  follows: 

1.  A  cover-glass  which  has  been  thoroughly  cleaned  is  held  in  a 
Stewart  or  similar  forceps. 

2.  Sterilize  the  platinum   loop  by  holding  it  over  the  flame  of   a 
Bunsen  burner  or  alcohol  lamp.    Allow  it  to  cool. 

3.  Dip  the  cool  platinum  loop  into  the  pus,  etc.,  and  spread  the 
drop  which  adheres  to  the  loop  in  a  thin  even  film  on  the  cover-glass. 
Sterilize  the  platinum  loop  again  and  put  it  aside. 

4.  Allow  the  cover-glass  to  become  air  dry,  then,  holding  it  in  the 
forceps,  draw  it,  with  the  prepared  side  upward,  three  times  through 


106  STAINING  OF  BACTERIA 

the  flame.  This  step  is  called  fixing  the  cover-glass.  By  exposing 
the  dried  pus,  containing  the  dried  bacteria,  to  the  heat  of  the  flame, 
the  proteids  (albumins)  are  coagulated,  and  the  spread,  with  its  pus 
corpuscles,  bacteria,  etc.,  now  adheres  firmly  to  the  cover-glass. 

5.  With  the  cover-glass  still  held  in  the  forceps,  stain  for  a  few 
minutes  with  one  of  the  above  watery  anilin  solutions  by  pouring 
the  stain  on  the  air-dried  fixed  cover-glass. 

6.  Wash  well  in  water  by  moving  the  cover-glass  about  in  the  fluid. 
Then  drop  it  on  filter  paper  and  dry  by  pressing  another  piece  of 
filter  paper  over  it.      Mount   on  a  slide  in  Canada   balsam   and 
examine  first  with  a  low-power  lens,  then  with  the  y1^  inch  (2  mm.) 
homogeneous  oil-immersion  lens.     To  mount  a  preparation,  put  the 
Canada  balsam  on  the  slide  and  drop  the  cover-glass,  prepared  side 
down,  on  the  balsam,  then  press  the  cover-glass  down. 

If  a  bacterial  cover-glass  preparation  from  a  pure  culture  is  to  be 
examined  the  first  steps  in  the  preparation  are  as  follows: 

1.  Clean  the  cover-glass.    Hold  it  in  a  Stewart  forceps,  and  with  a 
sterile  platinum  loop  place  a  small  drop  of  water  on  the  centre. 

2.  Hold  an  agar  or  gelatin  culture  tube  in  an  oblique  position 
between  the  index  and  middle  fingers  of  the  left  hand.    Remove  the 
cotton  plug  and  hold  it  between  the  middle  and  fourth  fingers  of  the 
left  hand.     Now  remove  from  the  open  culture  tube  with  the  sterile 
platinum  loop  a  very  small  amount  of  the  bacterial  growth  from  the 
surface  of  the  culture  medium.    Rub  up  with  the  platinum  loop  the 
growth  obtained  with  the  drop  of  water  on  the  cover-glass,  so  that 
an  even  emulsion  of  the  bacteria  is  formed.  ,  • 

3.  Allow  the  cover-glass  to  become  air  dry.    This  may  be  hastened, 
if  desired,  by  moving  it  rapidly  above  the  flame.     When  dry,  fix 
stain,  wash,  and  mount  as  above. 

Precautions  in  Working  with  Pathogenic  Bacteria. — In  working  with 
live,  highly  pathogenic  bacteria,  as,  for  instance,  glanders,  anthrax, 
tetanus,  etc.,  the  following  precautions  should  be  strictly  observed 
in  making  stained  cover-glass  preparations : 

1.  Have  on  the  table,  within  easy  reach  of  the  student,  a  large 
china  or  glass  vessel  (wooden  or  non-enamelled  metal  vessel  will  not 
do)  filled  with  a  strong  solution  of  bichloride  of  mercury  (corrosive 
sublimate)  at  least  1  to  1000,  better  still  stronger. 

2.  When  making  the  cover-glass  preparations  be  careful  not   to 
contaminate  anything;  sterilize  the  platinum  loop  well  before  laying  it 
down.    Never  hold  the  culture  tube  so  that  the  condensed  water  or 
the  bouillon  can  run  out  and  soil  the  hands,  table,  cotton  plug,  or 
anything  else. 

3.  Pour  the  stain  carefully  on  the  cover-glass,  so  that  none  of  it 
runs  over.     If  it  did  so  some  dangerous  pathogenic  bacteria  might 
be  washed  down  on  the  table. 

4.  After  the  stain  has  acted  long  enough,  pour  it  into  the  vessel 
containing  the  bichloride  solution.     In  washing  a  dangerous  prepara- 


WATERY  ANILIN  STAINS 


107 


FIG.  53 


Wash  bottle. 


tion  it  is  best  to  pour  some  water  from  a  small  bottle  or  a  so-called 
chemical  wash  bottle  over  the  preparation  so  that  the  washing  fluid 
may  run  directly  into  the  bichloride  solution. 

5.  After  being  washed,  drop  the  cover-glass  from  the  Stewart 
forceps  on  a  double  layer  of  filter  paper;  then  sterilize  the  end  of  the 
forceps  over  the  flame  of  the  Bunsen  burner. 
Now  put  aside  the  forceps  and  place  a  second 
double  layer  of  filter  paper  over  the  cover-glass 
and  dry  it  by  pressing  on  the  paper.  Pick  up 
the  cover-glass  carefully  at  the  margin  and 
move  it  about  a  little  over  the  flame  to  get  it 
perfectly  dry.  Finally,  mount  in  Canada 
balsam  and  throw  the  filter  paper  used  for 
drying  the  specimen  into  the  bichloride  solu- 
tion. 

The  student  should  not  imagine  that  staining 
with  watery  solutions,  air  drying,  and  fixing  in 
the  cold  kills  such  bacteria  and  their  spores, 
as  anthrax,  tetanus,  malignant  oedema,  etc. 
He  must,  therefore,  be  careful  with  his  cover- 
glass  preparation  until  it  is  safely  mounted  in 
the  Canada  balsam.  Some  bacteria  are  much 
more  dangerous  in  the  laboratory  than  out  in 

field  practice  among  patients.  The  glanders  bacillus  is  one  of  those 
which  has  killed  a  number  of  laboratory  workers,  hence  it  is  particu- 
larly necessary  to  be  careful  when  handling  it  in  pure  cultures. 

Watery  Anilin  Stains. — Besides  those  already  mentioned  the  fol- 
lowing watery  anilin  stains  are  frequently  used : 

LOEFFLER'S  ALKALINE  METHYLENE  BLUE.— 

Saturated  alcoholic  solution  of  methylene  blue 30  c.c. 

Watery  solution  of  caustic  potash  containing  the  latter  in  the  very  dilute  pro- 
portion of  1  to  10,000 100  c.c. 

This  is  an  excellent  all-around  dye.  It  stains  cover-glass  preparations 
in  from  three  to  five  minutes  or  in  even  less  time;  the  stain  does  not 
decompose  easily,  and  keeps  well  for  many  months  in  a  tightly  glass- 
stoppered  bottle. 

GRAM'S  METHOD  OF  STAINING  BACTERIA. — This  is  a  very  useful 
method,  because  it  permits  the  differentiation  of  certain  kinds  of 
bacteria,  which  are  morphologically  so  much  alike  that  they  could 
not  be  distinguished  merely  by  microscopic  examination.  The  fol- 
lowing solutions  are  necessary  for  this  stain : 

A.  Anilin-water  Gentian  Violet. — Take  about  nine  parts  of  dis- 
tilled water  and  one  part  of  anilin  oil.  Shake  well  in  a  flask  or  test- 
tube  and  filter  clear  through  an  ordinary  paper  filter.  Anilin  oil  is 
slightly  soluble  in  water.  In  a  saturated  solution  the  excess  of  the 
oil  shows  in  oily  droplets  in  the  fluid;  when  filtered  these  droplets  are 


108  STAINING  OF  BACTERIA 

arrested  by  the  paper  filter,  and  a  clear  watery  solution  with  the  smell 
of  anilin  oil  obtained.  This  watery  fluid  is  known  as  anilin  water. 
Add  to  the  latter  enough  of  a  saturated  alcoholic  solution  of  gentian 
violet  until  a  metallic  luster  is  produced  on  the  surface.  This  indi- 
cates that  the  gentian-violet  stain  has  been  added  to  the  point  of 
saturation.  The  strong  stain  which  has  now  been  prepared  is  known 
as  anilin-water  gentian  violet.  This  stain  does  not  keep  well  and  must 
be  made  fresh  every  few  days. 
B.  Grain's  Decolorizing  Fluid. — 

lodin 1  gram 

Iodide  of  potassium 2  grams 

Distilled  water 300  c.c. 

The  steps  in  staining  by  Gram's  method  are  as  follows: 

1.  Obtain  the  cover-glass  preparation  held  in  forceps,  from  pus, 
a  culture,  or  any  other  secretion  or  excretion,  as  already  described. 
Allow  it  to  become  air  dry  and  fix  in  the  flame  as  usual. 

2.  Cover   the   preparation   with   recently   prepared    anilin   water 
gentian  violet  and  allow  the  stain  to  act  for  several  minutes  in  the 
cold,  or,  better,  heat  slightly  over  a  flame. 

3.  Pour  off  the  stain  and  cover  with  Gram's  iodin  decolorizing  fluid; 
change  this  fluid  once,  and  allow  it  in  all  to  act  one  minute. 

4.  Pour  off  the  iodin  solution  and  wash  freely  in  95  per  cent, 
alcohol  until  no  more  violet  color  is  given  off. 

5.  Allow  the  alcohol  to  evaporate  or  dry  between  filter  paper,  and 
now  counter-stain  the  cover-glass  preparation  with  a  weak  watery 
solution  of  eosin  (J-  to  ^  watery  solution  of  eosin). 

6.  Dry  between  filter  paper,  mount  on  a  slide  in  Canada  balsam,  and 
examine  with  oil-immersion  lens.    Certain  bacteria,  if  treated  by  this 
method,  appear  in  a  deep  violet  color,  while  others  appear  in  a  faint 
eosin  (yellowish  pink)  stain.     The  bacteria  which  appear  in  violet 
are  said  to  be  stained  by  Gram's  method,  to  hold  the  Gram  stain, 
or  to  be  Gram  positive.     Those  which  are  stained  faintly  pink  do 
not  stain  by  Gram's  method,  lose  Gram's  stain,  or  are  Gram  negative. 

Gram  Positive  Bacteria. — The  following  are  some  of  the  important 
pathogenic  bacteria  which  are  Gram  positive  (appear  deep  violet  if 
stained  by  Gram's  method) : 

Staphylococcus  pyogenes  aureus,  albus,  and  citreus. 
Streptococcus  pyogenes.  Pneumococcus. 

Micrococcus  tetragenus.  Bacillus  diphtheria). 

Bacillus  tuberculosis.  Bacillus  anthracis. 

Bacillus  tetani.  Actinomyces. 

Bacillus  aerogenes  capsulatus. 

Gram  Negative  Bacteria. — The  following  are  some  of  the  impor- 
tant pathogenic  bacteria  which  are  Gram  negative  (appear  light 
pink  if  stained  by  Gram's  method) : 


STAINING  OF  CAPSULES  109 

Gonococcus.  Diplococcus  meningitidis. 

Diplococcus  catarrhalis.  Bacillus  typhosus. 

Spirillum  of  Asiatic  cholera.  Bacillus  coli  communis. 

Spirillum  of  fowl  cholera.  Bacillus  of  dysentery. 

Spirillum  of  Metchnikoff.  Bacillus  of  hog  cholera. 

Spirillum  of  Finkler  and  Prior.  Bacillus  of  influenza. 

Bacillus  of  black-leg.  Bacillus  of  glanders. 

Bacillus  necrophorus.  Bacillus  pyocyaneus. 

Bacillus  of  malignant  edema.  Bacillus  mucosus  capsulatus. 

Bacillus  of  pneumonia  (Friedliinder).  Bacillus  of  bubonic  plague. 
Spirochetae  of  relapsing  fever. 

Acid-fast  Bacteria. — There  is  a  group  of  bacteria  known  as  acid- 
fast  bacilli.  These  take  the  stain  with  great  difficulty,  but  hold  it 
even  in  the  presence  of  dilute  acids  after  they  have  once  been  stained. 
To  dye  them  it  is  necessary  to  prepare  a  very  strong  staining  solution 
and  combine  it  with  a  substance  which  acts  as  a  mordant.  This 
strong  staining  solution,  if  used  for  a  short  time,  must  be  boiling  hot; 
otherwise,  at  ordinary  or  incubator  temperatures  it  must  be  used  for 
thirty  to  sixty  minutes  or  longer. 

Ziehl's  Carbol-fuchsin. — The  staining  fluid  known  as  Ziehl's  carbol- 
fuchsin  is  used  in  staining  the  following  acid-fast  bacilli,  viz.,  the 
tubercle,  leprosy,  smegma,  Moeller's  grass,  and  Johne's  cattle  disease 
bacillus. 

ZiehPs  Carbol-fuchsin:  take 

1.  Basic  fuchsin  in  crystals          .      .      .'    ,.-      ...      .      .  1  gram 

2.  Absolute  alcohol 10  c.c. 

3.  Water ;V.      .      .      .      .  100  c.c. 

4.  Carbolic  acid  (95  per  cent.) 5  c.c. 

Dissolve  No.  1  in  No.  2  and  No.  4  in  No.  3  and  mix.  (For  the  use  of  this  stain  see 
under  tubercle  bacillus.) 

Some  other  special  stains  are  given  in  the  chapters  on  the  different 
pathogenic  bacteria  for  which  they  have  been  particularly  devised  or 
for  which  they  are  specially  valuable. 

The  following  are  special  methods  to  bring  out  differential  parts 
of  bacteria,  such  as  capsules,  spores,  flagella,  etc. 

Staining  of  Capsules. — Johne's  Method. — 

1.  Stain  cover-glass  in  a  warm  2  per  cent,  solution  of  gentian  violet 
for  one  to  two  minutes. 

2.  Wash  in  water. 

3.  Apply  1  to  2  per  cent,  watery  solution  of  acetic  acid  for  ten 
seconds. 

4.  Wash  in  water. 

5.  Examine  cover-glass  mounted  in  water — not  in  Canada  balsam. 
Friedldnder 's  Method.— 

1.  Apply  to  fixed  cover-glass  preparation  a  1   per    cent,  watery 
solution  of  acetic  acid  for  two  minutes. 

2.  Wash  in  water  and  dry  between  filter  paper. 

3.  Stain  with  anilin-water  gentian-violet  solution  for  a  few  seconds. 

4.  Wash  in  water,  dry  between  filter  paper,  and  mount  in  Canada 
balsam 


110  STAINING  OF  BACTERIA 

Ribbert's  Method  — 

1.  Stain  for  several  minutes  in  the  following  solution:  Prepare  a  hot 
saturated  watery  solution  of  dahlia,  add  to  100  c.c.,  50  c.c.  alcohol 
(95  per  cent.)  and  12.5  c.c.  glacial  acetic  acid. 

2.  Wash  in  water,  dry  between  filter  paper,  and  mount  in  Canada 
balsam. 

Welch's  Method.- 

1.  Prepare  and  fix  cover-glass  as  usual,  then  cover  the  film  with 
glacial  acetic  acid  for  a  few  seconds. 

2.  Blow  off  the  glacial  acetic  acid  and  replace  by    anilin-water 
gentian  violet.    This  must  be  poured  on  several  times  to  wash  off  all 
the  acetic  acid. 

3.  Wash  in  a  1  to  2  per  cent,  solution  of  chloride  of  sodium.    Mount 
in  this  fluid  (not  in  Canada  balsam)  and  examine. 

Staining  of  Spores. — It  has  previously  been  stated  that  spores,  in 
consequence  of  the  possession  of  a  very  tough,  tenacious  membrane, 
cannot  be  stained  by  the  ordinary  methods,  but  require  a  special 
technique.  The  following  are  some  of  the  methods  employed : 

1.  Prepare  and  fix  cover-glass,  as  usual. 

2.  Float  film  on  Ziehl's  carbol-fuchsin  solution  contained  in  a 
beaker.    Place  beaker  over  a  small  flame  or  on  a  water  bath  and  keep 
the  staining  solution  boiling  for  twenty  to  thirty  minutes. 

3.  Wash  in  water. 

4.  Decolorize  in  the  following  solution:  Alcohol,  2  parts;  1  per 
cent,  acetic  acid  in  water,  1  part.     Keep  on  washing  until  no  more 
red  stain  is  given  off.    The  bacteria  are  now  decolorized,  except  the 
spores,  which  are  stained  red. 

5.  Wash  in  water. 

6.  Mount  cover-glass  on  a  slide  in  water  and  examine  with  a  J- 
inch  dry  lens  to  see  whether  spores  are  really  stained  properly.     If 
this  is  the  case,  counter-stain  for  a  few  minutes  in  a  weak  watery 
solution  of  methylene  blue. 

7.  Wash  in  water,  dry  between  filter  paper,  mount  in  Canada 
balsam.    Result  of  procedure — spores  red,  remainder  of  bacilli  blue 

Moeller's  Method.— 

1.  Prepare  and  fix  cover-glass  as  usual,  then  cover  it  for    two 
minutes  with  chloroform,  which  removes  fatty  matter. 

2.  Wash  in  water. 

3.  Pour  on  cover-glass  a  5  per  cent,  solution  of  chromic  acid  for 
1J  to  2  minutes. 

4.  Stain  with  watery  fuchsin  solution  heated  over  a  small  flame  for 
one  minute. 

5.  Decolorize  in  5  per  cent,  sulphuric  acid  for  five  seconds. 

6.  Wash  in  water. 

7.  Counter-stain  in  dilute  watery  methylene-blue  solution  for  one- 
half  minute. 

8.  Wash  in  water,  dry  between  filter  paper,  mount  in  Canada 
balsam. 


STAINING  OF  FLAGELLA  HI 

Klein's  Method. — See  chapter  on  the  Anthrax  Bacillus. 

Staining  of  Flagella. — Flagella,  like  spores,  cannot  be  dyed  by  the 
ordinary  methods,  and  it  is  difficult  to  get  a  good  flagellar  stain.  It 
is  necessary  to  prepare  the  cover-glasses  in  a  special  manner.  They 
must  first  be  carefully  washed  successively  in  strong  mineral  acid, 
water,  alcohol,  and  ether,  so  that  they  are  absolutely  free  from  dirt, 
grease,  etc.  The  steps  in  the  preparation  of  the  clean  cover-glasses 
are  then  as  follows: 

1.  Place  six  clean  cover-glasses  in  a  row,  and  with  the  platinum 
loop  place  a  small  drop  of  water  on  each. 

2.  Inoculate  the  drop  on  cover-glass  No.  1  three  times  from  the 
margin  of  the  growth  of  a  young  agar  culture,  about  eighteen  hours 
old,  and  not  over  twenty-four  hours  old.     Mix  up  the  growth  well 
with  the  water  to  make  a  uniform  emulsion. 

3.  Now    inoculate   drop  on   cover-glass  No.  2  three  times   from 
emulsion  No.  1  (on  cover-glass  No.  1),  and  having  made  a  uniform 
emulsion  on  No.  2. 

4.  Inoculate  No.  3  three  times  from  No.  2,  and  so  on  until  all  of 
the  six  drops  have  been  inoculated. 

5.  Allow  the  six  cover-glasses  to  become  air  dry,  then  fix  in  the 
following  manner:    Do  not  pick  up  cover-glasses  with  forceps   but 
hold  in  the  right  hand  between  thumb  and  index  finger,  and  while 
in  this  position  fix  by  passing  three  times  through  a  flame.    In  this 
manner  the  temperature  sense  of  the  worker  will  prevent  an  over- 
heating of  the  cover-glasses  and  a  burning  of  the  delicate  flagella. 

Treat  and  stain  all  six  cover-glasses  by  one  of  the  following  methods, 
and  if  successful,  several  good  preparations  are  generally  obtained. 

Loeffler's  Method. — 1.  Apply  to  fixed  cover-glass  the  following 
mordant  :* 

20  per  cent,  watery  solution  of  tannic  acid,  prepared  by  heating.      .     100  c.c. 

Watery  solution  of  sulphate  of  iron,  saturated  in  cold 50  c.c. 

Saturated  alcoholic  solution  of  fuchsin 10  c.c. 

Use  the  mordant,  moderately  heated,  for  one-half  to  one  minute. 

2.  Remove  the  mordant  and  wash  well  in  water. 

3.  Wash  in  alcohol. 

4.  Stain  with  a  warm  anilin-water  gentian-violet  solution  to  which 
has  been  added  a  trace  of  a  very  dilute  caustic  soda  solution. 

5.  Wash  in  water,  dry  between  filter  paper,  and  mount  in  Canada 
balsam. 

Loeffler's  method  for  staining  flagella  was  the  first  one  used  and 
published.  It  is  a  difficult  one  on  account  of  the  varying  amounts 
of  alkali  which  must  be  added  to  the  gentian  stain.  The  following 
method  furnishes  better  and  more  uniform  results : 

1  The  word  mordant  means  a  preparation  which  will  so  act  upon  a  substance  that  the  latter 
will  take  a  stain  more  easily  and  hold  it  more  firmly — for  instance,  tannic  acid  or  sulphate  of 
iron  are  used  as  mordants  in  the  dyeing  of  wool  in  technical  establishments. 


112  STAINING  OF  BACTERIA 

Bunge's  Method. — 1.  Prepare  and  fix  cover-glasses   as  described 
under  the  Loeffler  method. 

2.  Use  the  following  mordant,  which  must  be  several  days  old : 

Concentrated  watery  solution  of  tannic  acid  .  .  ...  75  c.c. 
5  per  cent,  watery  solution  of  liquor  ferri  sesquichlorati  .  .  25  c.c. 
Concentrated  aqueous  solution  of  fuchsin  .  .  .  .'  .  .  10  c.c. 

Place  mordant  in  a  beaker,  float  cover-glasses,  prepared  side  down 
on  surface  of  mordant,  heat  over  a  small  flame  until  fluid  begins  to 
steam,  but  do  not  allow  it  to  boil. 

3.  Wash  well  in  distilled  water. 

4.  Stain  with  carbol-gentian-violet  solution,  which  is  heated  until 
it  steams  on  the  cover-glass. 

5.  Wash  in  water,  dry,  mount  in  Canada  balsam. 

Carbol  gentian  violet  is  prepared  like  carbol-fuchsin,  viz.,  take — 

Gentian  violet          1  gram 

Absolute  alcohol 10  c.c. 

Aquae  dest 100  c.c. 

Carbolic  acid 5  c.c. 

Carbol  fuchsin  may  be  used  instead  of  the  carbol  gentian  violet. 

Pitfield's  Method. — Prepare  the  following  two  solutions,  keep  them 
separate,  and  before  use  filter  and  mix. 

A.  Saturated  aqueous  solution  of  alum 100  c.c. 

Saturated  alcoholic  solution  gentian  violet 10  c.c. 

B.  Tannic  acid 10  grams 

Aquae  dest 100  c.c 

Mix  in  equal  proportions   before  use.       Cover  film  with  staining 
fluid,  heat  over  a  small  flame  until  the  stain  boils,  leave  on  for  one 
minute,  wash  well  in  water,  dry,  and  mount  in  Canada  balsam. 
The  following  is  not  a  real  staining  but  a  silver  impregnation  method  : 
Van  Ermengem's  Method  of  Silvering  Flagella. — 1.  Prepare  cover- 
glasses  as  already  indicated,  and  apply  the  following  mordant,  which 
may  be  used  hot  for  five  minutes  or  cold  for  thirty  minutes.    Take — 

20  per  cent,  watery  solution  of  tannic  acid  .....  60  c.c. 

2  per  cent,  watery  solution  of  osmic  acid      .      ...      .  30  c.c. 

Glacial  acetic  acid    .      .      .      .      .      .      .,,.-.      .'      .      .    4  to  5  drops 

2.  Wash  in  water. 

3.  Wash  in  alcohol. 

4.  Immerse  for  one  to  three  seconds  in  \  to  1  per  cent,  watery 
solution  of  nitrate  of  silver. 

5.  WTash  for  several  seconds  in  the  following  solution : 

Gallic  acid     .      .    '. •  .   .  ,      .      .     •.      .      .7  5  grams 

Tannic  acid  ....      .      .      .      .      ...      .      .      .      .  3  grams 

Acetate  of  sodium   .      ....    •  V     .            10  grams 

Distilled  water *• :  .V   .  •      .      .      .      .  350  c.c. 

6.  Immerse  again  in  the  \  to  1  per  cent,  watery  solution  of  nitrate 
of   silver   and   move  cover-glass,  held  in   forceps,  continually  until 
it  assumes  a  black  color. 

7.  Wash  well  in  water,  dry,  mount  in  Canada  balsam. 


WRIGHT'S  STAIN  OF  EOSINATE  OF  METHYLENE  BLUE     U3 

Staining  the  Polar  Bodies  or  Babes-Ernst  Granules. — Neisser's 
Method. — 1.  Prepare  and  fix  cover-glass  as  usual. 

2.  Stain  with  the  following  solution  for  one  to  three  seconds : 

Methylene  blue .  1  gram 

Absolute  alcohol *    .      .      .  20  c.c. 

Glacial  acetic  acid   .............  50  c.c. 

Distilled  water  .      .      .      ;      .'.   T    *»  "...    .      .      .      .      .      .  1000  c.c. 

3.  Wash  in  water. 

4.  Counter-stain  in  a  2  per  cent,  watery  solution  of  Bismarck- 
brown. 

5.  Wash  in  water,  dry,  and  mount  in  Canada  balsam.    Result  of 
the  stain — polar  bodies  blue,  other  parts  of  bacterium  light  brown. 

Piorkowski's  Method. — 1.  Prepare  cover-glass  as  usual  and  stain 
for  one-half  to  one  minute  in  Loeffler's  alkaline  methylene  blue. 

2.  Decolorize  in  alcohol  containing  3  per  cent,  hydrochloric  acid 
for  five  seconds. 

3.  Wash  rapidly  in  water. 

4.  Counter-stain  in  a  1  per  cent,  watery  solution  of  eosin,  very 
rapidly. 

5.  Dry  between  filter  paper  and  mount.    Result  of  the  procedure — 
polar  bodies  blue,  rest  of  bacterium  eosin  pink  or  yellow. 

Wright  Stain  of  Eosinate  of  Methylene  Blue. — This  stain  is  an  ex- 
ceedingly useful  one,  and  it  has  a  wide  field  of  application,  not  so 
much  for  staining  bacteria  from  pure  cultures,  as  for  bacteria  in  pus, 
and  particularly  for  protozoa,  such  as  malarial  plasmodia,  trypan- 
osomes,  piroplasmata,  and  for  blood  films  in  general.  It  is  a  modi- 
fication of  the  stains  of  Romanowsky  and  Leishman.  The  dye  can 
be  bought  ready  made  or  can  be  easily  prepared  as  follows  : 

1.  Prepare   in  a  flask  a  \  per  cent,  of  sodium  bicarbonate  in  water. 
Add,  after  complete  solution,  1  per  cent,  of  methylene-blue  in  sub- 
stance (either  one  of  the  following  three  preparations  of  Gruebler's 
may  be  used:  methylene-blue  BX,  Koch's  or  Ehrlich's  rectified). 

2.  Place  the  sodium  bicarbonate  methylene-blue  solution  in    the 
steam  sterilizer,  where  it  is  left  for  one  hour  after  the  steam  is  up. 
Then  remove  and  allow  to  cool. 

3.  Prepare  a  -fa  per  cent,  watery  solution  of  eosin  (yellowish  eosin 
of  Gruebler). 

4.  Place  the  methylene-blue  solution  in  a  large  flat  dish  and  add  the 
YQ-  per  cent,  eosin  solution  gradually,  stirring  constantly  with  a  glass 
rod.     Keep  this  up  until  the  mixture  assumes  a  purplish  color  and 
until  a  scum  with  a  yellowish  metallic  lustre  forms  on  the  surface 
and  a  finely  granular  black  precipitate  appears  in  the  suspension. 
This  will  generally  require  about  500  c.c.  of  the  -fa  per  cent,  eosin 
solution  to  100  c.c.  of  steamed  alkaline  methylene-blue    solution. 
After  the  precipitate  has  formed  the  fluid  is  run  through  a  dense 
paper  filter  and  the  precipitate  collected.      The  fluid  which  runs 
through  the  filter  is  of  no  further  use,  and  is  not  kept.      The  pre- 

8 


114  STAINING  OF  BACTERIA 

cipitate  on  the  filter,  however,  is  carefully  dried  in  the  incubator  or 
in  a  drying  oven  at  a  low  temperature.  It  is  then  scraped  off  the  paper 
filter  and  from  it  is  prepared  a  saturated  solution  in  C.  P.  methylic 
alcohol  (Merck's).  Three-tenths  of  a  gram  of  the  dry  stain  will 
saturate  100  c.c.  of  C.  P.  methylic  alcohol.  This  is  the  staining  fluid, 
ready  for  use.  It  is  very  permanent  in  character,  provided  that  it  is 
kept  in  a  tightly  glass-stoppered  bottle.  Care  must  be  taken  from  the 
start  that  the  solution  does  not  become  diluted  with  water,  hence 
the  bottle  in  which  it  is  prepared  must  have  been  washed  out  with 
methylic  alcohol  (not  with  water). 

The  stain  also  fixes  the  preparation,  and  it  is  used  as  follows: 

1.  When  thoroughly  air  dry  do  not  draw  through  flame.    Prepare 
cover-glasses  or  slides  as  usual. 

2.  Pour  undiluted  stain  from  glass-stoppered  bottle  on  the  cover- 
glass  or  slide  and  allow  it  to  act  for  one  minute. 

3.  Now  add  with  a  dropper,  drop  by  drop,  distilled  water  (dis- 
tilled water  must  be  used,  not  ordinary  tap  or  well  water)  until  the 
mixture  becomes  semitransparent,  with  a  reddish  tint  visible  at  its 
margins  and  a  metallic  scum  forms  on  the  surface.    The  amount  of 
water  required  will  vary  with  the  amount  of  staining  fluid  on  the 
preparation,  but  in  general  eight  or  ten  drops  will  be  sufficient  if  a 
seven-eighths  inch  square  cover-glass  is  used. 

4.  Wash  in  distilled  water  for  about  one-half  to  one  minute.    This 
will  so  differentiate  the  stain  that  nuclei  and  bacteria  appear  blue, 
while  cell  protoplasm  appears  pinkish.    (The  stain  also  differentiates 
well  neutrophilic,  eosinophilic,  and  basophilic  granula  of  the  various 
leukocytes.) 

5.  Dry  between  filter  paper  and  mount  in  Canada  balsam. 
Giemsa's  Stain  for  Spirochetse  and  Protozoa. — This  permanent  stain 

is  prepared  in  the  following  manner:  Take 

Azur  II,  eosin  (this  is  a  combination  stain)    ..•-.'..      .      .  •     3  grams 
Azur  II        .'.      .      .  _  .      .      .      .      ...      .      ...      .)     .    0.8  grams 

Dry  in  a  desiccator  over  sulphuric  acid,  powder  very  fine  and  sift 
through  a  very  fine  meshed  sieve.  Dissolve  in  C.  P.  glycerin  (Merck) 
250  c.c.  at  60°  C.  and  shake  continually.  Add  250  c.c.  Kahlbaum's 
C.  P.  methyl  alcohol  which  has  been  warmed  to  60°  C.  Keep  for 
twenty-four  hours  at  room  temperature  and  then  filter.  It  is  best  to 
buy  this  stain  ready  made  as  prepared  by  Gruebler,  in  Leipzig. 
Use  it  as  follows: 

1.  The  very  thin  cover-glass  or  slide  preparation  must  be  allowed 
to  become  air  dry. 

2.  Fix  for  fifteen  to  twenty  minutes  or  longer  in  absolute  alcohol. 

3.  Dilute  the  Giemsa  stain  as  follows:  Take  distilled  water  and 
add  to  each  cubic  centimeter  a  few  drops  of  a  y1^  per  cent,  solution 
of  carbonate  of  potassium.    To  each  cubic  centimeter  of  this  slightly 
alkaline  watery  solution  add  one  drop  of  the  Giemsa  stain. 


FIXING  OF  TISSUES  115 

4.  Use  on  fixed  cover-glass  or  slide  preparation  at  once  and  allow 
the  stain  to  act  not  less  than  one  hour,  better  several  hours. 

5.  Wash  well  in  water,  dry  carefully  between  filter  paper,  and 
mount  in  Canada  balsam. 

If  precipitates  have  been  formed  on  the  specimen  it  is  well  to 
wash  rapidly  a  few  seconds  in  90  per  cent,  alcohol  and  then  once 
more  stain  a  short  time  in  the  dilute  Giemsa  stain,  without  the  addi- 
tion of  carbonate  of  potash,  i.  e.,  a  drop  of  the  stain  to  each  cubic 
centimeter  of  pure  distilled  water. 

Blackening  of  the  Background  for  Demonstration  of  Fine  Spirochetse. 
— Freudenwald  has  published  recently  a  method  of  demonstrating  a 
few  fine  spirocheta?  (particularly  the  Spirochsetse  pallida)  in  secretions 
which  may  contain  them.  The  procedure  is  not  a  staining  method, 
but  one  in  which  a  dark  background  is  prepared  by  the  use  of  Chinese 
(India)  ink,  upon  which  the  microorganisms  appear  as  light  un- 
stained spiral  threads.  The  method  is  as  follows: 

1.  Take  a  platinum  loopful  of  the  discharge  which   is  suspected 
of  containing  the  microorganisms. 

2.  Mix  and  rub  it  up  well  with  a  drop  of  Chinese  ink  on  a  clean 
slide  (the  author  recommends  a  German  preparation  fluid  Chinese 
ink  of  Guenther  and  Wagner).     The  mixture  assumes  a  yellowish- 
brown  tint. 

3.  Spread  the  drop  out  into  a  thin  film  with  the  margin  of  another 
clean  slide  and  allow  it  to  become  air  dry. 

4.  The  preparation  can   now  be  directly  examined  with  the  oil- 
immersion  lens.    If  the  specimen  has  been  well  spread  in  a  thin  layer, 
the  spirochetae  appear  perfectly  white  on  a  yellowish  or  yellowish- 
brown  background.    After  examination  the  homogeneous  immersion 
oil  can  be  washed  off  with  xylol  and  the  preparation  can  be  pre- 
served as  a  permanent  one  for  future  use.     The  author  has  found 
this  new  method  very  simple  and  giving  very  excellent  results.     Any 
India  ink  may  be  used.     It  is  not  necessary  to  procure  the  German 
preparation  originally  recommended. 

Staining  Bacteria  in  Tissues. — It  is  often  desirable  and  necessary 
to  study  the  distribution  of  bacteria  in  tissues.  In  order  to  do  this 
successfully  the  tissues  must  first  be  properly  fixed,  embedded,  and 
sectioned. 

Fixing  of  Tissues. — Fixing  a  tissue  means  its  preservation  by  proper 
preserving  fluids  so  that  its  cells  and  other  elements  remain  as  nearly 
true  to  nature  as  possible.  Pieces  not  larger  than  one  cubic  centi- 
meter must  be  cut  out  with  a  sharp  scalpel  or  razor.  These  pieces  are 
dropped  at  once  into  strong  or  absolute  alcohol,  a  formalin  solution 
(1  part  of  formalin  to  nine  parts  of  water),  or  best,  if  certain  stains 
are  to  be  used,  into  Zenker's  solution. 

Bichromate  of  potassium ...     2.5  grams 

Sulphate  of  sodium       .  .      .     -.      .      .      .      .....      .     1.0  gram 

Corrosive  sublimate       .  .      ...      .      .......      .5.0  grams 

Water  .    100  c.c. 


116  STAINING  OF  BACTERIA 

Before  use  add  5  c.c.  of  glacial  acetic  acid.  The  fluid,  without  the 
acetic  acid,  may  be  made  up  in  bulk,  as  it  keeps  indefinitely,  but  the 
acid  can  only  be  added  shortly  before  use.  Leave  the  tissues  in  this 
fluid  for  from  two  to  twenty-four  hours,  according  to  the  size  of  the 
piece  and  the  hardness  or  softness  of  the  tissue.  Then  wash  for 
twenty-four  hours  in  running  water.  In  spite  of  this  washing  an 
insoluble  sulphite  of  mercury  will  remain  in  the  tissue  which  must  be 
removed  before  staining.  How  this  is  accomplished  with  the  aid  of 
Gram's  or  Lugol's  iodin  solution  is  described  under  the  steps  of  the 
eosin-methylene-blue  staining  method  following. 

After  tissues  have  been  fixed  in  a  watery  fluid,  or  in  95  per  cent, 
alcohol,  they  must  always  be  completely  dehydrated  in  absolute 
alcohol  or  some  other  suitable  medium.1  Only  after  this  has  been 
accomplished  can  the  tissues  be  embedded. 

Embedding  Methods. — There  are  two  principal  embedding  methods, 
and  the  object  of  both  is  to  get  the  tissue  into  such  shape  that  it  can 
be  cut  into  very  thin  sections  with  a  razor,  or,  what  is  much  better, 
a  machine  with  a  special  knife  called  a  microtome. 

A.  CELLOIDIN  EMBEDDING  METHOD. — 1.  After  fixation,  place  the 
tissue  into  absolute  alcohol  for  twenty-four  hours  and  change  the 
latter  once. 

2.  Place  into  equal  parts  of  absolute  alcohol  and  ether  for  one  day. 

3.  Place  in  thin  celloidin  at  least  for  a  day,  better  for  several  days. 

4.  Place  in  thick  celloidin  at  least  for  a  day,  better  for  several  days. 

5.  Paste  the  piece  of  tissue  with  thick  celloidin  on  a  block  of  wood, 
or,  better,  on  a  block  of  vulcanized  wood  fiber  (a  certain  wood  fiber 
material  impregnated  with  gutta-percha). 

6.  Allow  the  celloidin  on  the  block  and  on  the  tissue  to  become 
superficially  hard;  then  place  block  and  all  in  80  per  cent,  alcohol. 
After  twenty-four  hours   the  celloidin  has  hardened  well   and  the 
tissue  can  now  be  sectioned  with  the  microtome.     The  thin  and 
thick  celloidin  used  in  this  work  are  prepared  from  the  solid  imported 
celloidin,  which  comes  in  a  dark  bottle  put  up  with  water.      It  is 
prepared  for  use  as  follows: 

(a)  Drain  off  the  water  and  remove  all  traces  of  it  by  washing  in 
a  little  absolute  alcohol. 

(6)  Place  the  dry  celloidin  into  a  large  glass-stoppered  bottle,  cutting 
it  up  with  scissors  into  small  fragments  if  necessary.  Add  six  to 
eight  ounces  of  equal  parts  of  absolute  alcohol  and  ether  and  shake 
violently  until  all  the  celloidin  is  dissolved.  This  sometimes  takes  a 
couple  of  hours.  The  thick  syrupy  fluid  which  results  after  complete 
solution  of  the  celloidin  is  the  thick  celloidin.  Take  some  of  this  and 
dilute  it  with  ether  to  a  thin  consistency.  This  is  the  thin  celloidin. 
To  get  the  best  results  it  is  necessary  to  place  the  tissue  finally  in 
some  thick  celloidin  kept  in  a  Stender  or  Petri  dish  (see  Chapter  XII) 

i  Complete  dehydration  means  the  complete  abstraction  of  water. 


EMBEDDING  METHODS  117 

and  allow  the  alcohol  and  ether  to  evaporate  until  the  celloidin  has 
become  of  the  consistency  of  a  rather  soft  Swiss  cheese.  Then  the 
tissue  with  a  little  celloidin  around  it  can  be  cut  out  and  pasted  with 
a  little  thick  celloidin  on  the  fiber  block.  After  which  it  is  best  to 
leave  it  under  a  bell-jar  with  an  open  dish  of  chloroform.  In  this 
manner  the  most  homogeneous  embedding  best  adapted  for  section- 
ing is  obtained.  However,  it  is  not  necessary  in  ordinary  work  to 
adhere  strictly  to  all  of  these  finer  details. 

Celloidin  embedded  material  is  sectioned  on  the  microtome  in 
such  a  manner  that  the  microtome  knife  strikes  the  tissue  very  ob- 
liquely. Both  the  knife  and  tissues  should  be  kept  constantly  wet  with 
80  per  cent,  alcohol.  The  cut  sections  must  be  kept  in  80  per  cent, 
alcohol  until  they  can  be  stained. 

PARAFFIN  EMBEDDING  METHOD/ — 1.  Dehydrate  the  tissue  well 
in  absolute  alcohol. 

2.  Place  for  several  hours  in  equal  parts  of  absolute  alcohol  and 
xylol. 

3.  Place  for  several  hours  into  pure  xylol. 

4.  Place  for  several  hours  into  xylol  saturated  with  soft  paraffin. 

5.  Place  for  several  hours  in  melted  paraffin  kept  in  a  suitable 
paraffin  oven  at  about  54°  C. 

6.  Change  the  melted  paraffin  once  during  this  time. 

7.  Prepare  a  little  paper  box  or  place  two  lead  squares  on  a  glass 
plate,  pour  some  fresh  melted  paraffin  into  the  square  and  place 
the  tissue  in  it. 

8.  As  soon  as  the  paraffin  is  superficially  hard  place  the  tissue  with 
the  surrounding  paraffin  and  leads  into  the  refrigerator  or  into  cold 
water,  so  that  they  are  cooled  very  rapidly.    This  rapid  cooling  gives 
to  the  paraffin  a  homogeneous  consistency  which  makes  it  cut  better, 
and  much  thinner  sections  can  be  prepared.     When  the  tissue  and 
the  paraffin  have  been  thoroughly  cooled,   the  excess  paraffin  is 
trimmed  off,  leaving  the  tissue  surrounded  by  a  very  small  amount 
of  the  embedding  material.    The  embedded  tissue  is  then  mounted 
with  a  little  melted  paraffin  on  a  block  of  wood  or  vulcanized  wood 
fiber.    After  the  mounting  is  firm,  the  tissue  is  ready  to  be  sectioned 
on  the  microtome. 

Sectioning. — Paraffin  embedded  material  is  sectioned  dry  with 
the  knife  at  right  angles  to  the  block,  and  with  a  rapid  stroke.  Par- 
affin sections  that  are  to  be  stained  for  the  demonstration  of  bacteria 
should  be  very  thin — not  over  five  micra — and  should  lie  very  flat 
on  the  slide.  This  is  accomplished  by  taking  them  from  the  knife, 

1  A  very  rapid  paraffin-embedding  procedure  is  the  acetone  method,  which  has  the  following 
steps : 

1.  Place  small  tissue  into  best  water-free  pure  acetone  for  one  hour. 

2.  Change  acetone  and  leave  another  hour. 

3.  Drop  into  melted  paraffin  and  leave  in  paraffin  over  one-half  hour. 

4.  Change  paraffin  and  leave  another  half-hour.    The  tissue  is  now  ready  to  be  blocked  and 
sectioned. 


118 


STAINING  OF  BACTERIA 


and  floating  them  in  warm  water  (about  35°  to  40°  C.),  when  they 
flatten  out  perfectly.  The  next  step  is  to  prepare  a  clean  slide  by 
rubbing  over  it  a  small  amount  of  egg-albumen  mixture  composed  of 
equal  parts  of  beaten  white  of  egg  and  glycerin,  which  has  been 
filtered.  This  egg-albumen  glycerin  mixture  will  fix  the  section  on 
the  slide.  Dip  the  slide  into  the  warm  water  where  the  section  is 
floating,  and  guide  the  latter  with  a  platinum  rod  or  tissue  needle 
onto  the  slide  and  move  to  the  part  previously  prepared  with  the 
egg-albumen.  After  the  section  is  in  position  the  slide  is  placed  for 
several  hours  in  the  incubator  to  evaporate  the  water.  The  section 
now  rests  perfectly  flat  on  the  dry  slide. 

FIG.  54 


Paraffin  -embedding  oven. 

Removal  of  Paraffin. — It  is  now  necessary  to  fix  the  section  and 
remove  the  paraffin.  This  is  done  in  the  following  manner. 

1.  Move  the  slide  over  a  flame  (alcohol  lamp  or  small  flame  of 
Bunsen  burner)  until  the  paraffin  melts.  Then  heat  a  little  longer, 
so  that  the  egg-albumen  coagulates  and  fixes  the  section  on  the  slide. 
This  manipulation  requires  some  experience  which  can  be  gained 
only  by  practice.  If  heated  too  much  the  section  will  be  more  or 
less  damaged,  and  if  heated  too  little  there  is  great  danger  of  the 


STAINING  OF  SECTIONS  119 

section  floating  off   during   the  process   of  staining   in  the   watery 
solutions. 

2.  Place  the  section  in  xylol  which  will  dissolve  out  the  paraffin. 

3.  Next,  place  in  alcohol  which  will  wash  out  the  xylol. 

4.  Remove  the  alcohol  by  placing  the  section  in  water. 

FIG.  55 


Small  student's  microtome  for  sectioning  celloidin  or  paraffin-embedded  tissues. 

Staining  of  Sections. — The  section  is  now  ready  to  be  treated  by  one 
of  the  various  aniiin-staining  solutions  used  to  demonstrate  bacteria. 
It  is  frequently  advantageous  to  first  stain  the  section  by  some  of 
the  methods  used  in  normal  or  pathological  histology,  in  order  to 
bring  out  clearly  the  cellular  elements  of  the  tissue  itself.  If  this 
is  done  it  is  generally  best  to  stain  the  tissue  in  bulk  before  it  is 
embedded. 

Carmin  Stains. — Most  useful  for  such  staining  in  bulk  are  the 
carmin  stains,  particularly  alum  carmin,  which  is  prepared  as  follows: 

Carmin 2  grams 

Alum        .      .      .      . '  ; .    "  ...     .      .      .      .  5  grams 

Water      .      .    '.  ".      .'     .    ".      .      .      .    .  ^     ....        100  c.c. 

Boil  twenty  minutes,  then  add  enough  water  to  make  up  for  the  loss 
in  evaporation.  When  cool,  filter  and  add  a  crystal  of  thymol  to  pre- 
vent the  growth  of  moulds  in  the  staining  solution.  Tissues  to  be 
stained  in  bulk  should  be  left  in  this  carmin  solution  for  from  two  to 
three  days;  they  are  then  placed  for  one  to  two  hours  in  acid  alcohol 


120  STAINING  OF  BACTERIA 

(alcohol  70  per  cent.-— 100  c.c. — HC1 — 5  drops),  then  in  several 
changes  of  pure  alcohol,  so  that  every  trace  of  acid  is  removed,  and 
finally  they  are  embedded  in  celloidin  or  paraffin. 

Mallory's  Eosin-methylene  Blue  Stain. — When  staining  for  bac- 
teria in  tissues  it  is  frequently  necessary  to  employ  different  stains 
according  to  the  species  of  bacteria  to  be  demonstrated.  These 
stains  will,  therefore,  be  given  in  the  chapters  on  these  special  bac- 
teria. However,  there  is  one  method  of  staining  bacteria  known  as 
Mallory's  eosin-methylene  blue  stain,  which  has  a  wide  range  of  use- 
fulness and  which  furnishes  excellent  results.  To  get  the  best  results 
tissues  should  first  be  fixed  in  Zenker's  solution.  It  is  necessary  to 
remove  from  sections  so  fixed  the  precipitated  mercury  sulphite. 
The  steps  of  the  method  are  the  following: 

1.  After  removal  of  the  paraffin  and  xylol  from  the  sections,  place 
them  into  Gram's  decolorizing  fluid  (iodine  1  part,  iodide  of  potash 
2  parts,  water  300  parts)  for  twenty  minutes. 

2.  Wash  out  the  iodine  solution  in  95  per  cent,  alcohol  for  ten 
minutes. 

3.  Wash  in  water  and  stain  sections  for  twenty  to  thirty  minutes 
in  a  10  per  cent,  watery  eosin  solution. 

4.  Wash  rapidly  in  water  to  get  rid  of  the  excess  of  eosin. 

5.  Stain  in  Unna's  alkaline  methylene-blue  solution  diluted   with 
four  to  five  times  its  bulk  of  distilled  water  for  ten  to  fifteen  minutes. 
Formula  for  Unna's  alkaline  methylene-blue  solution: 

Methylene  blue  (Koch's) 1  gram 

Carbonate  of  potassium 1  gram 

Distilled  water 100  c.c. 

This  solution  keeps  several  months,  but  it  then  loses  in  staining 
power,  because  much  of  the  methylene  blue  is  oxidized  into  methyl 
violet  and  methylene  red. 

6.  Wash  in  water. 

7.  Wash,  for  the  purpose  of  decolorizing,  in  95  per  cent,  alcohol, 
keeping  the  slide  constantly  in  motion,  so  that  the  decolorizing  will 
go  on  uniformly.     When  the  pink  color  of  the  eosin  has  returned, 
dehydrate  in  absolute  alcohol   and  clear  in  xylol.     Then,  without 
mounting  in  Canada  balsam,  look  at  the  sections  with  the  low  power 
of  the  microscope.    If  the  nuclei  stand  out  well  differentiated  in  blue 
from  the  eosin-stained  protoplasm  the  section  has  been   decolorized 
enough.    If  there  is  still  too  much  blue  present  wash  in  95  per  cent, 
alcohol  again  until  the  differentiation  is  sufficient. 

8.  Finally  dry  and  mount  in  Canada  balsam  in  the  usual  manner. 
Gram's  Staining  Method  for  Paraffin  Sections. — 1.  Stain  section 

in  warm  anilin-water  gentian-violet  solution  for  twenty  minutes. 

2.  Wash  in  normal  salt  solution. 

3.  Decolorize  for  one  minute  in  Gram's   decolorizing   fluid  (iodin 
1  part,  iodide  of  potash  2  parts,  water  300  parts). 


STAINING  OF  SECTIONS  121 

4.  Wash  in  several  changes  of  absolute  alcohol  until  violet  color 
is  no  longer  given  off. 

5.  Clear  in  xylol  and  mount  in  Canada  balsam. 
Gram-Weigert  Method  for  Celloidin  Sections. — In  this,  as  in  the 

preceding  paraffin  section  method,  the  tissue  should  have  received 
a  preliminary  stain  in  carmin.  If  not  stained  in  bulk,  the  sections 
themselves  should  be  left  in  the  alum  carmin  over  night. 

1.  Fasten  the  celloidin  section  on  slide  with  ether  vapor  and  stain 
for  twenty  minutes  with  anilin-water  gentian  violet. 

2.  Wash  in  normal  salt  solution. 

3.  Leave  one  minute  in  the  iodin  solution  (solution  the  same  as 
in  preceding  method). 

4.  Wash  off  in  water. 

5.  Dry  with  filter  paper  to  remove  as  much  moisture  as  possible. 

6.  Wash  in  several  changes  of  anilin  oil  to  remove  most  of  the 
voilet  stain. 

7.  Clear  with  several  changes  of  xylol.    Examine  with  the  micro- 
scope before  mounting  in  Canada  balsam  to  see  whether  enough  of 
the  violet  stain  has  been  removed.    If  not,  wash  again  in  anilin  oil, 
then  in  xylol. 

8.  Finally,  mount  in  Canada  balsam. 

Levaditti  Silvering  Method  for  Exhibiting  Spirochetce,  particularly 
Spirochetce  pallida  in  Tissues  in  Congenital  Syphilis. — This  is  an 
impregnation  method  to  impregnate  microorganisms  in  tissues  with 
metallic  silver.  Although  not  a  staining  method,  it  should  be  con- 
sidered here.  The  steps  are  as  follows: 

1.  Fix  small  pieces  of  tissue  in  a  solution  of  formalin  1  part,  water 
3  parts  (10  per  cent,  formalin  solution)  for  twenty-four  hours. 

2.  In  95  per  cent,  alcohol  for  twenty-four  hours. 

3.  Wash  in  distilled  water. 

4.  Place  in  a  1^  per  cent,  solution  of  nitrate  of  silver  in  a  dark 
bottle  to  protect  against  light;  keep  in  the  incubator  for  three  days. 

5.  Wash  in  distilled  water. 

6.  Place  in  the  following  reducing  solution  (keep  in  light  and  at 
room  temperature): 

Pyrogallic  acid 2  grams 

Formalin .      .      .      .      .•     .          5  c.c. 

Distilled  water 100  c.c. 

7.  Wash  in  distilled  water. 

8.  Embed  in  celloidin  or  paraffin. 

9.  Dehydrate  sections,  clear  in  xylol,  and  without  staining  mount 
in  Canada  balsam. 

The  spirochetae  appear  perfectly  black  on  a  yellowish  background. 


122  STAINING  OF  BACTERIA 


QUESTIONS. 

1.  What  is  an  anilin  stain?    What  three  types  are  distinguished? 

2.  Name  the  three  anilin  stains  most  commonly  employed  in  working  with 
bacteria.    How  are  the  solutions  of  these  stains  prepared? 

3.  Describe  the   method  of  staining  a  cover-glass  preparation  of  pus  for 
bacteria. 

4.  Describe  method  of  preparing  and  staining  a  cover-glass  specimen  from  a 
pure  culture. 

5.  What  precautionary  measure  should  be  observed  when  staining  live  patho- 
genic bacteria? 

6.  Why  is  it  particularly  dangerous  to  work  with  live  cultures  of  the  glanders 
bacillus? 

7.  Give  formula  for  Loeffler's  alkaline  methylene  blue. 

8.  Give  formula  for  (a)  anilin-water  gentian  violet;  (6)  Gram's  decolorizing 
fluid. 

9.  Describe  Gram's  method  of  staining  and  decolorizing  bacteria. 

10.  Name  some  bacteria  which  stain  by  Gram's  method  and  some  which  do 
not. 

11.  Give  formula  for  Ziehl's  carbol-fuchsin. 

12.  Give  Johne's  method  for  staining  the  capsules  of  bacteria. 

13.  Describe  one  of  the  methods  for  staining  spores. 

14.  Give  one  of  the  methods  for  staining  flagella. 

15.  Describe  the  method  of  staining  the  polar  bodies. 

16.  Describe  the  method  of  using  Wright's  stain  on  a  cover-glass  preparation 
of  blood  or  pus. 

17.  Describe  the  use  of  Giemsa's  stain  for  demonstrating  Spirochsetse  pallida. 

18.  Describe  the  celloidin -embedding  method. 

19.  Describe  the  paraffin-embedding  method. 

20.  Describe  Mallory's  eosin-methylene-blue  staining  method. 

21.  Describe  the  Gram-Weigert  staining  method  for  tissues. 

22.  Describe  Levaditti's  silvering  method. 

NOTE. — The  student  need  not  spend  much  time  in  learning  by  heart  formulae 
for  stains  and  staining  methods;  these  are  best  acquired  by  practice. 


CHAPTER    X. 

CULTURE  MEDIA  AND  THEIR  STERILIZATION. 

A  SUCCESSFUL  investigation  of  pathogenic  bacteria,  permitting  of 
definite,  trustworthy  conclusions,  is  possible  only  if  they  can  be 
obtained  in  pure  culture.  By  this  term  is  meant  the  isolation  of  one 
species  of  bacterium  to  the  exclusion  of  all  other  living  organisms.  A 
pure  culture,  accordingly,  represents  an  otherwise  sterile  culture 
medium  containing  only  one  species  of  microorganisms.  To  isolate 
pathogenic  bacteria  suitable  sterile  culture  media  are  needed.  Some 
of  these  substances,  such  as  blood  serum,  milk,  potatoes,  occur  in 
nature  and  are  simply  sterilized  by  suitable  methods  without,  as  a 
rule,  adding  anything  to  them;  others  are  artificially  compounded  from 
various  substances  and  chemicals.  In  the  preparation  of  artificial 
culture  media,  the  few  necessary  ingredients  must  be  so  selected  and 
mixed  in  proper  proportions  that  they  supply  all  the  elements  neces- 
sary for  the  growth  and  multiplication  of  bacteria.  At  the  same  time 
the  medium  must  be  either  neutral  or  faintly  alkaline.  Pathogenic 
bacteria  generally  grow  best  on  a  faintly  alkaline  medium;  a  few  also 
grow  on  a  very  slightly  acid  soil,  but  the  latter  is  rather  exceptional. 
Substances  which  are  changed  in  a  characteristic  manner  by  certain 
bacteria  are  frequently  added.  Sugar,  for  instance,  is  introduced  to 
show  whether  the  bacterium  which  is  being  grown  possesses  the 
faculty  of  splitting  up  sugar  and  forming  carbon  dioxide.  Litmus  in 
an  alkaline  medium  indicates  whether  the  growing  bacterium  forms 
acids  and  finally  changes  the  alkaline  reaction  to  acid. 

Natural  sterilized  culture  media,  such  as  milk,  coagulated  blood 
serum,  potatoes,  etc.,  are  not  transparent.  Very  often,  however,  it  is 
desirable  to  work  with  perfectly  transparent  culture  media.  These  can 
be  obtained  by  adding  to  suitable  clear  solutions  either  gelatin  or  a  sub- 
stance called  agar-agar,  derived  from  a  Japanese  sea- weed.  Both  these 
substances,  when  dissolved  in  a  watery  fluid  by  heat,  form  transparent 
masses  with  it.  Gelatin  culture  media  which  melt  at  about  25°  C. 
cannot  be  kept  in  the  incubator  without  losing  their  solid  consistency. 
Generally,  as  stated,  culture  media  must  be  sterilized.  Sometimes, 
however,  it  is  desirable  to  use  natural  media,  such  as  blood,  blood 
serum,  ascitic  fluid,  etc.,  without  subjecting  them  to  heat.  In  such 
cases  the  fluids  must  be  obtained  in  a  perfectly  aseptic  manner,  so 
that  they  are  and  remain  sterile. 

Sterile  blood  serum  may  be  obtained  from  the  living  animal. 
Since  the  method  in  the  case  of  culture  media  is  identical  with  that 


124  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

employed  in  the  collection  of  an  antitoxic  or  immune  serum,  it  may  be 
here  described. 

Method  of  Obtaining  Sterile  Blood  Serum  from  a  Horse. — 1.  Restrain 
the  horse  so  that  it  can  be  readily  manipulated  and  cannot  disturb 
the  operator. 

2.  Shave  a  few  square  inches  of  skin  a  little  above  the  middle  of 
the  jugular  vein. 

3.  Sterilize  the  skin  by  scrubbing  well  with  soap  and  water,  then 
with   alcohol,  next  with   solution  (1  to  500  to  1000)  of  bichlorid  of 
mercury,  and  finally  with   sterile  distilled   water.      (An   alternate 
method  consists  in  shaving  the  skin  on  the  previous  day  and  applying 
a  10  per  cent,  alcoholic  solution  of  iodin  to  the  entirely  dry  skin,  half 
an  hour  before  the  operation.)    Cover  the  sterilized  skin  with  sterile 
cotton. 

4.  Have  ready  a  large,  sterile  hypodermic  needle  or  small  curved 
trocar  connected  with  a  small   rubber  tube,  leading  into  a  sterile, 
cotton-stoppered,  cylindrical  glass  vessel.    The  entire  apparatus  must 
be  sterile. 

5.  When  the  preparations  are  completed  the  operator  must  sterilize 
his  hands  as  carefully  as  for  an  important  aseptic  operation.     An 
assistant  then  compresses  the  jugular  vein  in  the  lower  portion  of 
the  neck.     After  removal  of  the  sterile  cotton  from  the  previously 
shaved  and   sterilized  skin  the   trocar  is   pushed   into  the  jugular 
vein  and  the  blood  flows  through  it  into  the  sterile  receptacle. 

6.  When  a  sufficient  quantity  has  been  collected  it  is  placed  on 
ice  for  twenty-four  to  forty-eight  hours  and  the  serum  is  then  de- 
canted off  from  the  coagulum.    Too  much  stress  cannot  be  laid  upon 
the  importance  of  performing  every  step  in  an  absolutely  aseptic 
manner. 

Sterilization  of  Bacterial  Culture  Media. — This  is  generally  accom- 
plished by  means  of  steam  heat,  generated  in  a  suitable  vessel  called 
a  steam  sterilizer.  In  the  ordinary  apparatus  the  steam  passes  out 
without  obstruction;  in  others  it  is  retained  under  a  pressure  of 
several  atmospheres  and  the  temperature  rises  above  100°  C.  Those 
of  the  latter  type  are  called  autoclaves.  Sterilization  is  completed  in 
them  in  a  much  shorter  time  (about  one  hour)  than  in  the  common  ster- 
ilizers with  free  streaming  steam.  When  culture  media  are  subjected 
in  the  steam  sterilizer  to  the  temperature  of  boiling  water  (100°  C.) 
for  a  period  of  three  to  four  hours  or  longer,  all  bacteria  and  their 
spores  are  destroyed.  This  method  is  called  continuous  sterilization. 

Certain  media,  like  blood  serum  or  gelatin,  however,  are  rendered 
worthless  by  this  method.  The  former  becomes  a  grumous,  broken-up 
mass,  the  latter  turns  into  a  permanent  fluid  and  refuses  to  coagulate 
again.  Continuous  sterilization  in  the  ordinary  steam  sterilizer  or 
the  autoclave  cannot,  therefore,  be  employed  in  the  case  of  these 
substances  or  transudates  like  ascitic  fluid,  etc.,  but  short  periods  of 
discontinuous  heating  will  answer  the  purpose.  This  method  is 


STERILIZATION  OF  BLOOD  SERUM 


125 


known  as  the  fractional  sterilization  of  Tyndall.  Its  principle  is  as 
follows:  When  culture  media  are  heated  for  a  short  time  at  a  higher 
temperature  (for  example  for  thirty  minutes  at  60°  to  100°  C.),  all 
adult  vegatative  forms  of  bacteria  are  destroyed.  Many  spores, 
however,  survive  this  treat- 
ment. If,  after  having  been  FIG.  57 
heated,  the  medium  is  left  in 
a  warm  place  for  twenty-four 
hours,  most  of  the  remaining 
live  spores  will  develop  into 
vegetative  forms.  These  are 
killed  by  another  short  expos- 
ure to  heat.  Several  repeti- 
tions of  this  process  enable 
all  the  spores  to  develop  and 
bring  about  the  destruction 
of  all  the  bacteria.  Culture 
media  containing  gelatin  are, 

FIG.  56 


Arnold  steam  sterilizer. 


Autoclave,  or  digester,  used  for  sterilizing 
under  pressure. 


therefore,  sterilized  by  placing  them  on  three  or  four  consecutive 
days  in  the  steam  sterilizer  for  periods  of  from  twenty  to  thirty  min- 
utes, and  keeping  them  in  the  intervals  in  a  warm  place. 

Sterilization  of  Blood  Serum  or  Transudates  Containing  Blood  Plasma. — 
This  is  accomplished  in  the  following  manner:  The  fluid  is  collected 


126  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

with  all  possible  aseptic  precautions  and  distributed  to  sterile  test- 
tubes.  In  these  it  is  heated  on  a  water  bath  or  a  special  blood-serum 
sterilizing  and  coagulating  apparatus  (Robert  Koch)  for  five  to  seven 
consecutive  days  for  one  hour  at  60°  C.  During  the  intervals  it  is 
always  kept  in  a  warm  place.  On  the  seventh  or  eighth  day  it  is 
treated  as  usual  and  then  the  temperature  is  slowly  raised  to  90°  C. 
and  maintained  at  this  height  until  the  serum  in  the  test-tubes  is 
firmly  coagulated.  The  tubes,  while  in  the  Koch  apparatus,  are 
kept  in  a  slanting  position.  After  removal  from  the  apparatus  they 
are  preserved  in  a  cool  place  until  needed.  Blood  serum  may  be  ob- 
tained from  sheep,  cattle,  or  swine,  at  a  slaughter  house,  or  it  may  be 
drawn  directly  from  the  horse,  sheep,  or  goat.  In  the  former  case  it 
should  be  collected  in  sterile  glass  receptacles  when  the  bloodvessels 
of  the  neck  are  cut  and  placed  in  the  refrigerator  with  as  little  delay 
as  possible.  The  less  these  vessels  are  shaken  the  clearer  a  serum 
may  be  expected.  After  having  been  on  ice  for  from  twenty-four  to 
forty-eight  hours  the  serum  has  generally  well  separated  from  the 
clot  or  coagulum.  The  former  may  now  be  poured  or  pipetted 
off  and  distributed  into  sterile  test-tubes  or  other  sterile  glass 
receptacles. 

FIG.  58  FIG.  59 


Blood-serum  coagulator.  .      Dry-heat  sterUizer. 

Sterilization  of  Glassware. — All  glassware  used  for  culture  media 
must  be  sterilized  before  use.  This  is  best  accomplished  in  a  dry-air 
sterilizer.  The  apparatus  is  constructed  on  the  principle  of  a  baking 
oven  as  found  in  an  ordinary  kitchen  gas  stove  or  as  a  detached 
kitchen  utensil.  In  fact,  a  baking  oven  of  this  kind  may  be  used 
as  a  dry  sterilizer.  In  bacteriological  work  the  temperature  for  dry 
sterilization  may  be  raised  to  160°  to  200°  C.  Because  new  glassware 
often  contains  a  slight  deposit  of  soluble  alkalies,  test-tubes,  flasks, 
etc.,  must  be  soaked  in  water  acidulated  with  hydrochloric,  nitric,  or 
sulphuric  acid  before  being  used  for  the  first  time  and  afterward 
washed  in  pure  water  until  every  trace  of  acid  has  been  removed; 
otherwise  the  alkalies  would  subsequently  enter  the  culture  media 


PROTECTION  AGAINST  EVAPORATION  127 

and  might  change  their  reaction  sufficiently  to  interfere  with  bacterial 
growth. 

Flasks,  test-tubes,  etc.,  before  being  sterilized  in  the  hot-air  steril- 
izer must  be  stoppered  by  plugs  of  clean  cotton.  A  good  surgical 
cotton  gives  the  best  service.  Pieces  of  it  are  rolled  up  fairly  tightly 
into  a  conical  plug,  which  is  forced  into  the  mouth,  so  that  some  of 
the  cotton  projects  in  mushroom-shape  over  the  rim  of  the  glass. 
When  the  test-tubes  are  to  be  filled  with  culture  media  the  cotton 
plug  is  removed  and  afterward  replaced.  The  filled  tubes  are  then 
sterilized  by  one  of  the  methods  adapted  for  the  particular  medium. 

It  is,  of  course,  evident  that  some  air  will  enter  the  tubes  through 
the  cotton  after  they  are  taken  out  of  the  sterilizer  and  cooled.  Air 
contains  microorganisms,  but  since  it  is  filtered  through  the  dense 
mass  of  cotton  all  bacteria  are  efficiently  prevented  from  entering. 
It  is  particularly  necessary  to  sterilize  all  glassware  in  which  blood 
serum,  blood-serum  mixtures,  gelatin  and  gelatin  mixtures  are  to  be 
kept,  because  these  media  must  be  sterilized  by  fractional  sterili- 
zation and  this  method  is  not  as  safe  as  a  long  continuous  or  autoclave 
sterilization. 

Filling  of  Test-tubes. — The  best  method  for  filling  test-tubes 
with  agar  or  gelatin  is"  as  follows:  A  piece  of  rubber  tubing  is 
connected  with  the  stem  of  a  glass  funnel  and  the  free  end  of  the 
hose  is  provided  with  a  glass  tube  drawn  out  into  a  narrow  outlet; 
the  hose  should  also  have  a  burette  clamp.  The  melted  agar  or 
gelatin  is  poured  en  masse  into  the  funnel  which  has  previously  been 
warmed  with  hot  water  and  small  amounts  can  be  let  out  conveniently 
into  each  test-tube.  A  special  filling  apparatus  has  also  been  devised. 
It  is  constructed  somewhat  like  a  chemical  separatory  funnel  and 
from  it  a  definite  amount  of  the  melted  medium  (5  or  10  c.c.)  can  be 
let  out  into  the  test-tubes.  In  filling  test-tubes,  it  is  important  not  to 
allow  any  of  the  medium  to.  adhere  to  the  mouth  of  the  tube,  as  this 
would  make  the  cotton  plug  stick  to  the  tube. 

Protection  against  Evaporation. — When  media  distributed  in  test- 
tubes  are  to  be  kept  for  some  time  or  are  to  be  placed  in  the  incubator 
for  days  and  weeks,  they  must  be  protected  against  evaporation. 
For  this  purpose  rubber  caps  are  used.  Before  being  applied  to  the 
mouth  of  the  test-tube  the  caps  must  be  soaked  for  a  number  of  hours 
in  a  solution  of  bichlorid  of  mercury  (1  to  500  or  1000).  This  kills 
the  bacteria  and  moulds  which  might  adhere  to  the  rubber,  and  by 
growing  through  the  moist  cotton,  drop  into  the  culture  medium,  thus 
contaminating  it.  Another  method  is  to  seal  the  mouth  of  the  test-tube 
with  paraffin  or  sealing  wax  after  the  cotton  has  been  pushed  down 
to  some  extent.  Perhaps  the  best  method,  of  preserving  culture  media 
in  test-tubes  for  a  longer  period  of  time,  and  yet  having  them  always  ready 
and  easily  accessible,  is  the  following:  Take  an  anatomical  jar  of  the 
Whitall-Tatum  type  or  an  ordinary  Mason  jar,  place  some  cotton 
on  the  bottom,  moisten  with  a  strong  formalin  solution,  introduce 


128  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

the  tubes  so  that  they  stand  in  an  upright  position,  moisten  the  rubber 
ring  with  formalin  solution  and  then  screw  on  the  lid.  Prepared  in 
this  manner  culture  tubes  may  be  kept  for  many  months.  Gelatin 
tubes,  however,  if  kept  in  this  fashion  for  a  long  time  may  lose  their 
property  of  being  liquefied  by  liquefying  bacteria. 

FIG.  60  FIG.  61 


Erlenmeyer  flask.  Pasteur  flask. 

Preparation  of  Nutrient  Bouillon  from  Fresh  Meat. — The  basis  of 
most  solid  artificial  culture  media  in  use  is  nutrient  bouillon.  This 
in  itself  forms  an  excellent  culture  soil  for  the  majority  of  patho- 
genic bacteria.  On  account  of  the  great  advantages  offered  by 
solid  culture  media  in  isolating  bacteria  in  pure  cultures,  bouillon  is 
frequently  combined  with  agar-agar  or  gelatin.  Nutrient  bouillon 
may  be  prepared  by  either  one  of  two  methods.  Take  one  pound 
(500  grams)  of  finely  chopped,  lean,  boneless  meat  (generally  beef, 
for  special  purposes  veal,  pork,  horse  or  dog  meat),  add  two  quarts 
(1000  c.c.)  of  water,  and  allow  it  to  stand  for  twelve  to  eighteen 
hours  in  the  refrigerator  or  in  winter  in  the  cold.  Filter  through 
muslin  and  thoroughly  express  the  juice  which  remains  in  the 
meat.  Boil  to  precipitate  the  coagulable  albumins,  then  filter  and 
make  up  to  1000  c.c.  Add  10  grams  of  dried  beef  peptone  and  5 
grams  of  common  salt  (NaCl)  to  the  filtrate.  Boil  again  until  it  is 
entirely  dissolved.  Test  the  reaction  with  litmus  paper.  It  will  be 
found  to  be  acid.  Add  enough  of  a  solution  of  sodium  hydrate  to 
make  the  bouillon  very  slightly  alkaline  to  litmus.  •  Boil  again  and 
filter  clear.  Distribute  into  test-tubes,  about  10  c.c.  in  each,  or  small 
flasks,  about  50  to  100  c.c.  in  each.  Sterilize  by  continuous  sterili- 
zation in  the  ordinary  steam  sterilizer  or  autoclave.  It  is  sometimes, 
though  not  generally,  necessary  to  procure  culture  media  of  a  very 
definite  reaction.  In  this  case  the  bouillon  is  titrated  with  a  one- 
twentieth  normal  solution  of  caustic  soda  or  sodium  hydrate  (NaOH). 
A  small  portion  of  the  uncorrected  bouillon  is  taken,  diluted  with 
nine  times  the  amount  of  distilled  water,  and  titrated  exactly  with 
the  Y¥  sodium  hydrate  solution  until  neutral  or  faintly  acid  to  a 
phenolphthalein  reaction  indicator.  The  amount  of  the  caustic  soda 


PREPARATION  OF  NUTRIENT  BOUILLON  FROM  FRESH  MEAT   129 


solution  necessary  to  neutralize  100  c.c.  of  the  bouillon  is  then  cal- 
culated and  the  calculated  amount  of  a  normal  solution  of  sodium 
hydrate,  less  1.5  or  1  c.c.  is  added  for  each  100  c.c.  of  the  bouillon. 


FIG.  62 


FIG.  63 


Mohr's  burette  and  clamp. 
FIG.  64 


Mohr's  burette  and  holder. 
FIG.  65 


Burette  clamps. 


130  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

Such  a  bouillon  is  designated  as  a  +  1.5  or  +  1.0  bouillon.  Its 
reaction  is  acid  toward  the  phenolphthalein  indicator  but  alkaline 
toward  litmus. 

An  easier  method  for  preparing  the  nutrient  bouillon  is  the  fol- 
lowing: Take  4  to  6  grams  of  meat  extract,  10  grams  of  dried  beef 
peptone,  5  grams  of  common  salt,  dissolve  in  enough  water  to  make 
1000  c.c.  Boil  well  and  neutralize  with  caustic  soda  solution,  then 
filter  clear,  distribute  to  test-tubes,  and  sterilize  as  before. 

Gelatin. — 1.  Prepare  1000  c.c.  of  a  clear,  faintly  alkaline  nutrient 
bouillon.  Heat. 

2.  Dissolve  100  grams  of  best  clear  French  gelatin  in  the  hot  solu- 
tion.   This  again  makes  the  solution  quite  acid. 

3.  Add  caustic  soda  solution  until  the  mixture  becomes  faintly 
alkaline  to  litmus. 

4.  Prepare  a  funnel  with  a  double  paper  filter  through  which 
boiling  hot  water  has  been  poured.    When  the  latter  has  drained  off, 
filter  the  gelatin  through  the  hot  filter  into  a  flask.     This  must  be 
done  in  a  warm  room,  as  otherwise  the  filter  is  likely  to  cool,  and  the 
gelatin  may  set  in  it.     (If  necessary,  redissolve  the  gelatin  over  a 
water  bath,  not  over  an  open  flame.) 

5.  While  still  warm,  distribute  the  filtered  gelatin  into  sterile  test- 
tubes,  about  10  c.c.  to  each. 

6.  Sterilize  by  fractional  sterilization,  as  already  described,  for  three 
or  four  days  in  the  steam  sterilizer  (not  the  autoclave).    During  the 
intervals  keep  in  a  warm  place,  but  the  temperature  must  not  be  so 
high  as  to  cause  the  gelatin  to  remain  fluid. 

7.  After  the  last  sterilization  in  the  steam  sterilizer,  keep  the  tubes 
in  an  upright  position  so  that  the  gelatin  sets  in  a  cylindrical  mass 
(not  with  a  slanting  surface). 

A  good  gelatin  culture  media  must  be  perfectly  transparent. 
Agar-agar. — 1.  Prepare  1000  c.c.  of  clear,  faintly  alkaline,  nutrient 
bouillon. 

2.  Take  15  to  20  grams  of  agar-agar  and  cut  the  long  strips  into 
small  pieces,  the  smaller  the  better.    Soak  for  from  twelve  to  twenty- 
four  hours  in  cold  water.    Drain  the  water  off  through  a  cloth  and 
wring  out   the  swollen   mass,  so  as  to  remove  as   much  water  as 
possible  out  of  the  agar-agar. 

3.  Add  the  agar-agar  to  the  bouillon  and  heat  until  the  former  is 
entirely  dissolved.    The  heating  may  be  carried  on  in  an  autoclave, 
a  steam  sterilizer,  or  over  an  open  flame.    In  the  latter  case  the  mix- 
ture must  be  stirred  continually  so  that  the  agar-agar  does  not  burn. 

4.  When  solution  is  completed,  test  the  reaction  again.     As  a  rule 
it  does,  not  change,  but  remains  slightly  alkaline,  as  it  was  originally. 

5.  Filter  clear.    This  is  the  most  difficult  and  tedious  process  in 
the  preparation  of  a  good  agar.    Filtration  is  very  slow  and  sometimes 
requires  a  number  of  days.    The  process  must  be  carried  on  in  the 
autoclave,  the  steam  sterilizer,  or  through  a  double  jacketed  copper 


AGAR-AGAR 


131 


FIG.  66 


filter,  which  can  be  filled  with  water  and  kept  hot  over  a  Bunsen 
burner  (with  a  ring-shaped  burner  with  many  small  openings).  When 
it  is  required  on  short  notice  a  tolerably  good  agar  can  be  prepared  by 
sedimentation,  as  follows:  After  the  agar  is  entirely  dissolved  it  is 
placed  in  cylindrical  vessels,  which  are  thoroughly  heated  in  the 
steam  sterilizer  and  left  there  when  the  flame  is  turned  off.  During 
the  very  slow  cooling  of  the  agar  the  impurities  fall  to  the  bottom  of 
the  vessel.  When  entirely  cold  the  agar  is  caked  out  and  the  lower 
stratum  containing  the  impurities  cut  off  and  thrown  away. 

6.  After  removal  of  the  impurities  by  filtration  or  sedimentation 
the  agar  is  remelted  and  distributed  into  test-tubes,  about  10  c.c.  to 
each.  It  is  then  sterilized  by  con- 
tinuous sterilization  in  the  autoclave 
or  steam  sterilizer,  and  taken  out 
while  hot  and  fluid.  The  tubes  are 
placed  on  an  inclined  plane,  so  that 
the  agar  solidifies  in  a  slanting 
position.  This  yields  the  largest 
possible  surface  for  inoculation. 

A  good  2  per  cent,  agar,  the 
preparation  of  which  has  been  de- 
scribed above,  should  be  free  from 
impurities  and  transparent,  but  it 
is  never  as  entirely  clear  as  a  first- 
class  gelatin  medium.  Two  per 
cent,  agar  melts  at  80°  C.,  and  re- 
solidifies at  40°  C.  It  can,  therefore, 
be  kept  in  the  incubator  without 
melting.  Ten  per  cent,  gelatin 
melting  at  25°  C.  cannot  be  kept  in 
the  incubator.  The  transparency 
and  brilliancy  of  both  gelatin  and 
agar  may  be  improved  by  the 
addition  of  the  white  of  an  egg  before  filtration.  The  egg  is  broken 
and  the  white  separated  from  the  yolk.  The  albumen  is  mixed  with 
water  and  gradually  added  under  constant  stirring  to  the  melted 
gelatin  or  agar,  the  temperature  of  which,  however,  must  not  exceed 
60°  C.  in  order  to  avoid  coagulation  of  the  egg-albumen.  After  thor- 
oughly mixing  the  media  are  heated  in  the  steam  sterilizer  or  on  a 
water  bath.  The  egg-albumen  coagulates  and  carries  with  it  to  the 
bottom  fine  impurities,  difficult  to  filter  out  without  the  coagulated 
albumen. 

Both  gelatin  and  agar  frequently  receive  certain  additions  for 
special  purposes.  Such  additions  may  also  be  made  to  blood  serum 
or  the  latter  may  be  combined  with  agar.  The  following  culture 
media  are  frequently  used  in  determining  definite  characteristics  of 
certain  bacteria  when  raised  in  pure  culture. 


Double-walled  hot-water  funnel  with  cir- 
cular gas  burner  and  stand. 


132  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

Sugar  Gelatin  or  Sugar  Agar. — This  is  prepared  like  the  ordinary 
gelatin  or  agar  plus  the  addition  of  generally  1  to  2  per  cent, 
glucose,  lactose,  maltose,  saccharose,  etc. 


FIG.  67 


Adjustable  copper  trays  for  slanting  agar  or  blood-serum  tubes. 


Agar  slants  ready  for  use. 

Glucose  Formate  Gelatin  (Kitasato). — Ordinary  gelatin  plus  2  per 
cent,  glucose  and  0.4  per  cent,  of  sodium  formate. 

Litmus  Gelatin. — Ordinary  nutrient  gelatin  plus  a  sufficient  quan- 
tity of  sterile  litmus  solution  to  give  it  a  lavender  color. 

Lactose  Litmus  Gelatin. — Ordinary  nutrient  gelatin  plus  2  per  cent, 
lactose  plus  a  sufficient  quantity  of  sterile  litmus  solution  to  give  the 
transparent  culture  medium  a  pale  lavender  color. 

Glycerin  Agar. — Prepare  nutrient  bouillon  as  usual,  but  add  finally 
50  c.c.  of  pure  neutral  glycerin  for  each  1000  c.c.;  then  treat  the  agar 
as  described  above. 

Sugar  Agar. — Nutrient  agar  plus  2  per  cent,  glucose,  lactose,  maltose, 
saccharose,  etc. 


BLOOD  AGAR 


133 


Lactose  Litmus  Agar. — Ordinary  nutrient  agar  plus  2  per  cent, 
lactose  and  a  sufficient  quantity  of  litmus  solution  to  give  to  the 
medium  a  pale  lavender  color. 

Gelatin  Agar. — This  is  prepared  like  the  ordinary  nutrient  gelatin. 
It  contains  both  gelatin  and  agar  in  the  following  proportions:  If  it 
is  to  be  incubated  at  30°  C.,  gelatin,  10  per  cent.;  agar,  0.5  per  cent.; 
if  it  is  to  be  incubated  at  37°  C.,  gelatin,  12  per  cent.;  agar  0.75  per 
cent.  The  gelatin  and  the  agar  are  dissolved  separately  in  propor- 
tionate amounts  of  the  nutrient  bouillon.  After  separate  filtration 
and  clearing  they  are  mixed  and  sterilized  by  the  fractional  method, 
as  described  for  nutrient  gelatin. 

Loe filer's  Blood-serum  Mixture. — Prepare  an  ordinary  nutrient 
bouillon  (veal  will  give  better  results  than  beef)  and,  before  correcting 
the  reaction  and  sterilization,  add  1  per  cent,  glucose.  To  each  100 
c.c.  of  the  sterile  bouillon  add  300  c.c.  of  blood  serum,  distribute  the 
mixture  to  sterile  test-tubes  and  sterilize  according  to  the  method 
given  for  blood  serum.  Coagulate  the  mixture  in  a  slanting  position. 


FIG.  69 


FIG.  70 


Wire  baskets  for  agar  or  gelatin  tubes. 

Serum  Bouillon. — Collect  some  ascitic,  pleuritic,  or  hydrocele  fluid 
with  aseptic  precautions  in  a  sterile  flask.  Mix  the  serous  fluid  with 
twice  its  bulk  of  sterile  nutrient  bouillon.  Distribute  to  sterile  test- 
tubes,  sterilize  by  the  fractional,  discontinuous  method  for  a  week, 
but  do  not  finally  raise  the  temperature  to  the  coagulation  point  of 
albumin. 

Blood  Agar. — Prepare  nutrient  agar  by  the  usual  method  and  keep 
the  tubes  in  the  incubator  for  a  few  days  to  permit  the  condensed 
water  to  evaporate.  Obtain  blood  from  a  rabbit  with  aseptic  pre- 
cautions by  exposing  the  jugular  and  drawing  it  into  a  sterile  all- 
glass  syringe  or  a  glass  pipette.  Squirt  the  blood  into  a  small  sterile 
flask  containing  pieces  of  glass  or  glass  pearls.  Defibrinate  the  blood 
by  shaking,  and  pour  a  small  amount  of  the  fluid  mixture  of  blood 
serum  and  corpuscles  into  the  dry  agar  tubes,  which  can  be  used  at 
once.  In  spite  of  all  precautionary  measures  a  certain  number  of 
tubes  will  generally  be  contaminated.  Pigeon's  blood  is  also  used. 


134  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

Hemoglobin  Agar. — Prepare  agar  tubes  by  the  usual  method  and 
keep  them  in  the  incubator  for  some  time  to  permit  the  condensed 
water  to  evaporate.  Prepare  hemoglobin  as  follows :  Allow  aseptically 
obtained  blood  to  run  into  a  flask  containing  sterile  physiologic  salt 
solution.  Shake  and  leave  in  the  refrigerator  until  the  red  blood 
corpuscles  have  settled.  Pipette  off  the  clear  fluid  and  replace  by 
fresh  sterile  salt  solution.  Repeat  this  operation  once.  (The  pro- 
cedure has  been  fully  described  above  as  the  washing  of  red  blood  cor- 
puscles). If  only  a  small  amount  of  hemoglobin  is  needed  the  washing 
may  be  done  in  the  tubes  of  the  centrifuge  in  fifteen  minutes.  Remove 
the  hemoglobin  from  the  corpuscles  by  shaking  with  ether;  evaporate 
the  latter  on  a  water  bath  at  a  low  temperature  or  in  the  incubator, 
and  filter  the  watery  solution  of  hemoglobin  through  a  Pasteur  filter. 
Add  the  clear  hemoglobin  filtrate  to  dry  sterile  agar  tubes. 

Milk. — Milk  is  sometimes  used  as  a  culture  medium.  It  is  first 
boiled,  the  cream  is  removed  after  it  has  separated  out,  and  10  c.c. 
of  the  fat-free  milk  is  placed  into  test-tubes.  These  are  then  sterilized 
as  usual.  Before  the  tubes  are  prepared  the  reaction  of  the  milk  must 
be  tested;  it  should  be  •+  10  to  +  20  acid  to  phenolphthalein,  but 
neutral  or  faintly  alkaline  to  litmus. 

Litmus  Milk. — Prepare  the  milk  as  above  and  before  distributing 
it  to  tubes  add  enough  sterile  litmus  solution  to  give  it  a  deep  lavender 
color.  If  the  milk  is  too  acid  to  produce  this  color  effect,  add  enough 
-£$  solution  of  caustic  soda  to  produce  the  desired  color. 

Beer  Wort.— -Wort  gelatin  and  wort  agar  are  frequently  used  for 
the  cultivation  of  yeast  cells,  saccharomyces,  or  blastomyces.  Beer 
wort,  before  hops  have  been  added,  may  be  obtained  from  a  brewery. 
It  can  also  be  prepared  in  a  laboratory  as  follows: 

1.  Take  250  grams  of  crushed  malt  and  place  it  in  a  2  liter  flask. 

2.  Add  1000  c.c.  distilled  water  heated  to  70°  C.  and  close  the  flask 
with  a  rubber  stopper. 

3.  Place  in  a  water  bath  kept  at  60°  C.  for  one  hour. 

4.  Strain  through  muslin  into  another  flask  and  heat  in  the  steam 
sterilizer  for  one-half  hour. 

5.  Filter  through  a  dense  paper  filter  and  sterilize  in  the  steam 
sterilizer.     If  the  beer  wort  is  to  be  used  as  such  it  should  be  dis- 
tributed to  the  test-tubes  before  the  sterilization. 

Wort  Gelatin  and  Wort  Agar. — These  are  prepared  like  ordinary 
nutrient  gelatin  or  agar,  except  that  the  nutrient  bouillon  is  replaced 
by  beer  wort.  The  reaction  of  the  latter  is  not  interfered  with; 
however,  if  gelatin  is  used  the  strongly  acid  reaction  of  the  latter  has 
to  be  corrected. 

Potato  Culture  Media. — For  ordinary  work  the  preparation  is  as 
follows:  Select  good  potatoes  which  have  not  yet  germinated  in  the 
cellar;  potatoes  which  have  been  frozen  cannot  be  used.  Their  out- 
side must  be  thoroughly  cleansed  by  scrubbing  with  soap  and  water, 
then  with  pure  water,  and  finally  with  1  to  1000  bichloride  solution. 


SPECIAL  CULTURE  MEDIA  135 

This  operation  is  necessary  because  earth  bacteria  often  adhere  to 
the  outer  surface  and  it  is  desirable  to  get  rid  of  their  exceedingly 
resistant  spores  which  can  withstand  steam  sterilization  for  more  than 
four  hours.  Cut  out  the  eyes,  and,  after  peeling,  cut  the  potatoes  into 
slices,  subsequently  to  be  placed  in  Petri  dishes  (see  Chapter  XII)  and 
into  cylinders  which  are  divided  into  two  equal  masses  to  be  placed  in 
test-tubes.  Cylinders  are  prepared  with  an  ordinary  household  apple 
corer  or  with  a  special  device,  the  Ravenel  potato  cutter.  The  slices 
and  cylinders  are  then  best  washed  in  running  water  for  several  hours 
to  insure  partly  against  subsequent  undesirable  changes.  After  wash- 
ing, the  material  is  placed  into  the  glass  receptacles.  The  tubes  first 
receive  a  piece  of  broken  glass  or  some  glass  wool  on  which  the  half 
cylinder  rests,  and  a  little  distilled  water  to  supply  moisture.  The 
potatoes  then  undergo  fractional  or  continuous  sterilization.  In 
spite  of  all  precautions,  potato  media  frequently  dry  out  and  become 
dark  during  preservation  or  later  in  the  incubator.  Special  points 
in  the  use  of  potatoes  are  mentioned  under  anthrax  and  glanders  (see 
Chapters  on  these  Bacteria). 

Special  Culture  Media. — Certain  growing  bacteria  have  definite 
biologic  characteristics,  such  as  the  production  of  indol,  etc.,  and  a 
number  of  special  culture  media  are  used  to  determine  these.  Other 
special  media  are  free  from  albumins  or  proteid  matter,  and  contain 
chemicals  of  well-known  formula?  only.  Some  media  of  this  type  are: 

Dunham's  Peptone  Water. — 1.  Witte's  dry 'pep tone,  10  grams; 
common  salt,  5  grams;  mix  with  250  c.c.  of  distilled  water,  heated  to 
60°  C. 

2.  Place  into  a  flask  and  make  up  with  distilled  water  to  1000  c.c. 

3.  Heat  to  boiling  for  one-half  hour. 

4.  Cool  and  filter. 

5.  Distribute  to  test-tubes  and  sterilize  in  the  steam  sterilizer  for 
twenty  minutes  on  three  consecutive  days. 

Peptone  Rosolic  Acid  Water. — 1.  Take  Dunham's  peptone  water, 
100  c.c.,  and  add  2  c.c.  of  J  per  cent,  alcoholic  solution  of  rosolic 
acid  (coralline). 

2.  Heat  for  one-half  hour. 

3.  Filter  through  filter  paper. 

4.  Distribute  to  test-tubes  and  sterilize  as  above. 

Nitrate  Water. — 1.  Dissolve  10  grams  of  peptone  in  1000  c.c.  of 
ammonia-free  distilled  water. 

2.  Heat  in  steam  sterilizer  for  twenty  minutes. 

3.  Add  1  gram  of  sodium  nitrite  (C.  P.). 

4.  Filter,  distribute  to  tubes,  and  sterilize  as  above. 
Albumin-free  Solutions. — Pasteur's  Solution. 

Ammonium  tartrate    ......' 1  part 

Saccharose  or  cane  sugar 10  parts 

Yeast-ash 1  part 

Water    .      . 100  parts 


136  CULTURE  MEDIA  AND  THEIR  STERILIZATION 

Cohn's  Solution. 

Phosphate  of  potash 0.10  gram 

Crystals  of  sulphate  of  magnesium    . 0.10  gram 

Tribasic  phosphate  of  calcium      .    '  .  ;    ..  ,  .      .      .      .      .        0.01  gram 
Ammonium  tartrate     .      .      .      .      .   '  .      .'....'.      .        0.20  gram 

Aq.  dest.       .      .      ....      .      .  v  .      .      .      .      ,      .  20.00  c.c. 

Ushinsky's  Solution. 

Chloride  of  sodium       .      .  .      .      .      .      .      ....  5  to  7  grams 

Chloride  of  calcium       .      .  '.      ...      .  ^    .      .      .  0.1  gram 

Sulphate  of  magnesium      .  .      ...      ....    0.2  to  0.4  gram 

Dipotassium  phosphate     .  .      .      .      .      ....      .       2  to  2.5  grams 

Ammonium  lactate  (acid) 6.7  gram 

Asparaginate  of  sodium    .  .      .      .      .      .      .      .      .  3.5  gram 

Dissolve  in  1000  c.c.  of  distilled  water  and  add  30  to  40  c.c.  of 
glycerin.    Sterilize  by  the  discontinuous  method. 
Fraenkel's  Solution. 

Chloride  of  sodium        .      .      .'...*.      .      .  .  5  grams 

Potassium  diphosphate •;.-.  .  2  grams 

Ammonium  lactate  (neutral)        .      .'   .      .      .      .      .  .  6  grams 

Commercial  asparagin        .      .      .      .      .      .      ...      .  .  4  grams 

Aq.  dest .      .      .      .....  .  1000  c.c. 

Add  enough  caustic  soda  solution  to  make  the   reaction    faintly 
alkaline,  distribute  to  tubes  and  sterilize  by  the  discontinuous  method. 
Winogradsky's  Solution  for  Nitrifying  Microorganisms. 

Sulphate  of  ammonium 1  gram 

Sulphate  of  potassium 1  gram 

Dissolve  in  distilled  water  1000  c.c.  and  add  a  previously  sterilized 
solution  of  magnesium  carbonate.  Distribute  to  tubes  or  flasks  and 
sterilize  for  three  days  by  fractional  method  for  periods  of  twenty 
minutes  each. 

Winogradsky's  Culture  Medium  for  Bacteria  which  Oxidize  Nitrites 
into  Nitrates. 

Sodium  nitrite,  C.  P.  /.  .      .      .      .      .      ....'.  1.0  gram 

Potassium  phosphate  . 0.5  gram 

Magnesium  sulphate  ...      ...      .  ,  .      .      .      .      „  0.3  gram 

Carbonate  of  soda  (dry)  .      .      .      .     f      ..     .      .      ...  1.0  gram 

Chloride  of  sodium   .  . .      .      .  0.5  gram 

Sulphate  of  iron        .  .  .....      .      .      ....  0.4  gram 

Distilled  water    .      *.  .  ...      ....      ...      .'  1000  c.c. 

This  fluid  also  forms  the  base  of  a  1.5  per  cent,  agar  medium. 
Nitrate  Solution. 

Sodium  chloride,  C.  P V    .      .         0.5  gram 

Dry  peptone .      .w    *      .      .      •>-          1.0  gram 

Potassium  nitrate,  C.  P.      .      .      .      .      .      .     '.      .      .      .          0.2  gram 

Distilled  water    .  .......  1000  c.c 


QUESTIONS  137 


QUESTIONS. 

1.  What  do  we  mean  by  the  term  pure  culture? 

2.  What  is  a  culture  medium? 

3.  What  is  a  natural,  what  an  artificial  culture  medium? 

4.  What  are  the  requirements  of  a  culture  medium  on  which  bacteria  can  be 
grown?    What  should  be  the  reaction  of  a  culture  medium? 

5.  What  substances  are  used  to  prepare  solid  transparent  culture  media? 

6.  At  what  temperatures  do  the  ordinary  gelatin  and  agar  culture  media  melt  ? 

7.  Describe  the  method  of  obtaining  sterile  blood  serum  from  a  horse. 

8.  How  are  the  artificial  culture  media  generally  sterilized  ? 

9.  What  is  meant  by  the  term  sterilization  ? 

10.  What  is  an  Arnold  steam  sterilizer? 

11.  What  is  an  autoclave? 

12.  What  is  meant  by  continuous  sterilization? 

13.  What  is  TyndalPs  fractional  sterilization  method?    What  is  its  underlying 
principle  ? 

14.  How  is  agar,  how  is  gelatin  sterilized,  and  why? 

15.  How  is  blood  serum  in  tubes  sterilized  and  why? 

16.  How  is  the  blood  for  use  as  a  bacterial  culture  medium  generally  obtained 
and  how  treated  afterward? 

17.  How  is  the  glassware  used  for  work  in  bacteriology  sterilized? 

18.  How  should  new  glassware  be  treated  before  use  in  work  with  bacteria  and 
why? 

19.  Give  the  formula  for  the  preparation  of  the  ordinary  nutrient  bouillon. 

20.  What  is  meant  by  a  +1.5  bouillon?    How  does  it  act  toward  the  phenol- 
phthalein,  how  toward  the  litmus  indicator? 

21.  What  is  the  meaning  of  the  word  indicator  as  used  in  chemistry? 

22.  What  is  the  meaning  of  the  word  normal,  or  standard,  or  molecular  solution 
in  chemistry? 

23.  How  is  a  T^  (one-tenth)  normal  solution  of  NaHO  (caustic  soda)  prepared? 

24.  Give  the  formula  for  preparing  the  ordinary  gelatin  as  used  in  the  bac- 
teriologic  laboratory. 

25.  Give  the  same  formula  for  agar. 

26.  How  is  agar  filtered  or  cleared? 

27.  Name  some  substances  frequently  added  to  the  ordinary  culture  media. 
Why  added? 

28.  What  is  a  glucose  gelatin,  what  a  lactose  litmus  gelatin,  what  a  glycerin 
agar,  what  a  litmus  milk? 

29.  How  is  Loeffler's  blood  serum  mixture  prepared? 

N.  B. — The  student  should  only  attempt  to  commit  to  memory  the  formulae 
for  the  most  commonly  used  artificial  culture  media,  such  as  ordinary  gelatin 
and  agar  and  those  indicated  in  questions  28  and  29.  It  is  unnecessary  to  commit 
to  memory  all  of  the  formulas  given  above. 


CHAPTER    XL 

CULTURE  MEDIA  IN  RELATION  TO  METABOLIC  PRODUCTS- 
TESTS  FOR  THE  LATTER. 

A  CONSIDERABLE  number  of  the  culture  media  described  in  the 
preceding  chapter  are  especially  devised  in  order  to  demonstrate  the 
formation  of  certain  metabolic  products  of  bacterial  growth,  which 
in  some  instances  are  so  characteristic  that  they  assist  materially  in 
the  identification  of  certain  species  of  bacteria. 

Proteolytic  Ferments. — As  already  pointed  out,  it  is  often  important 
to  determine  whether  bacteria  produce  a  peptonizing,  proteolytic 
ferment.  This  can  be  ascertained  by  growing  the  culture  on  the 
boiled  white  of  an  egg,  blood  serum,  or  gelatin.  Since  the  liquefaction 
of  gelatin  is  the  most  complete,  it  is  the  preferable  medium  for  the 
identification  of  peptonizing  species.  The  presence  of  other  ferments, 
such  as  diastase,  invertin,  etc,  sometimes  has  to  be  determined. 

Diastase. — The  enzyme  diastase  can  be  demonstrated  by  the  addi- 
tion of  starch  to  a  fluid  culture  medium  which  has  been  made  abso- 
lutely sugar-free.  To  remove  the  traces  of  sugar  contained  in  both 
meat  and  meat  extract  the  former  after  having  been  finely  chopped 
is  kept  for  two  days  at  10°  to  15°  C.  At  this  temperature  the  muscle 
sugar  is  decomposed  into  lactic  acid.  According  to  Theobald  Smith, 
the  meat  of  poorly  nourished  tubercular  cattle  is  also  free  from  sugar. 
A  sugar-free  bouillon  can  also  be  prepared  by  inoculating  nutrient 
bouillon  with  colon  bacilli  (which  decompose  the  sugar)  and  subse- 
quent sterilization  and  filtration  of  the  medium. 

After  a  sugar-free  bouillon  has  been  obtained,  from  1  to  2  per  cent, 
of  the  best  acid-free  starch  is  added  and  gelatinized.  The  process 
is  as  follows:  Shake  together  200  c.c.  sugar-free  bouillon  and  10  to 
20  grams  of  starch;  heat  800  c.c.  of  bouillon  to  the  boiling  point  and 
add  gradually  the  200  c.c.  of  starch  emulsion.  Stir  continually  to 
insure  a  uniform  gelatinizing  of  the  starch,  distribute  to  test-tubes  and 
flasks  and  sterilize  by  the  fractional  method.  Long-continued  sterili- 
zation might  form  sugar  from  the  starch.  After  sterilization  some 
of  the  tubes  and  flasks  are  tested  for  sugar,  and  if  free  from  it  the 
media  may  be  inoculated.  If  a  diastase  or  amylolytic  ferment  is  liber- 
ated in  the  bacterial  growth  some  of  the  starch  will  be  converted  into 
amylodextrin,  achroodextrin,  and  maltose.  The  presence  of  these 
bodies  is  indicated  (1)  by  a  liquefaction  of  the  gelatinized  starch, 
(2)  by  the  iodin  test,  (3)  by  testing  the  reducing  power  of  the 
fluid  with  Fehling's  solution,  the  well-known  reagent  determining 


ACID  FORMATION  AND  ALCOHOL  139 

the  presence  of  sugars.  The  iodin  test  is  made  by  adding  and 
mixing  successively  a  few  drops  of  Gram's  iodine  solution  with 
the  media.  Amylodextrin  and  erythrodextrin,  if  present,  produce  a 
purple  color;  erythrodextrin  and  achroodextrin,  a  port-wine  color; 
achroodextrin  and  maltose,  no  coloration.  The  qualitative  test  for 
maltose  is  made  with* Fehl ing's  solution  in  the  usual  manner:  Take  a 
few  cubic  centimeters  of  Fehling's  solution,  prepared  from  equal 
amounts  of  solution  No.  1  and  No.  2,  dilute  with  distilled  water,  heat 
to  boiling,  and  then  add,  drop  by  drop,  some  of  the  previously  diluted 
and  filtered  starch  culture  medium.  If  maltose  is  present  an  orange 
or  red-brown  precipitate  will  be  formed.  If  the  sugar  formed  is  to 
be  determined  quantitatively  it  must  first  be  ascertained  whether 
it  is  maltose  or  dextrose.  This  can  be  done  by  changing  the  sugar 
present  into  an  ozazon  by  the  action  of  phenylhydrazin  hydrochlorate 
and  acetate  of  potash  and  determining  the  form  of  crystallization, 
the  melting  point,  and  the  amount  of  nitrogen  present  in  the  com- 
pound. These  are  somewhat  more  complicated,  though  not  difficult, 
chemical  manipulations,  the  details  of  which  can  be  found  in  a 
text-book  on  organic  quantitative  analyses.  After  the  kind  of  sugar 
present  has  been  found  the  amount  can  also  be  ascertained  by 
titrating  with  Fehling's  solution. 

Invertin. — To  demonstrate  the  formation  of  invertin  in  a  bacterial 
growth  the  latter  must  be  inoculated  into  a  sugar-free  bouillon  to 
which  a  small  amount  of  pure  saccharose  or  cane  sugar,  which  does 
not  possess  any  reducing  power,  finally  has  been  added.  The  presence 
of  invertase  is  manifested  by  the  inversion  of  some  or  all  of  the  cane 
sugar  into  invert  sugar  (dextrose,  maltose,  etc.),  which  reduces 
Fehling's  solution. 

Rennet  and  "Lab"  Enzymes. — The  presence  of  rennet  and  "lab" 
enzymes  is  ascertained  in  the  following  manner :  Prepare  a  sugar-free 
bouillon  and  inoculate  several  tubes  with  the  bacterium  to  be  tested. 
After  keeping  the  growth  a  few  days  in  the  incubator,  heat  the  tubes 
for  thirty  minutes  in  a  water  bath  at  55°  C.  This  will  destroy  the  bac- 
teria but  not  the  rennet  if  any  is  present.  After  heating,  add  about 
5  c.c.  of  the  contents  of  these  tubes  to  sterile  litmus  milk  in  another 
set  of  tubes.  Keep  for  several  days  at  22°  C.,  and  examine  every 
day  for  ten  or  twelve  days.  If  there  is  no  coagulation  at  the  end  of 
this  time  rennet  is  not  present. 

Acid  Formation  and  Alcohol. — Acid  formation  by  bacteria  is  accu- 
rately determined  by  inoculating  them  into  500  to  1000  c.c.  of  sterile 
sugar  bouillon  (see  above)  of  a  known  definite  slightly  alkaline  re- 
action. After  a  number  of  days  the  reaction  of  the  inoculated  bouillon 
is  again  titrated  by  the  aid  of  a  one-twentieth  normal  solution  of 
caustic  soda.  The  difference  in  the  results  of  the  two  estimations 
indicates  the  amount  of  acid  formed.  If  the  growth  in  the  flask  is 
very  heavy  it  may  be  necessary  to  filter  the  bouillon  through  a  Pasteur 
or  Berkefeld  filter  before  the  final  tests  can  be  made. 


140     CULTURE  MEDIA  IN  RELATION  TO  METABOLIC  PRODUCTS 

If  alcohol  has  been  formed  from  the  sugar  it  will  be  necessary  to 
obtain  it  by  fractional  distillation.  The  details  of  exact  acid  and 
alcohol  determinations  require  a  more  complicated  apparatus  and 
extensive  set  of  reagents,  hence  they  cannot  be  given  here.  The 
mere  fact  of  acid  formation  can  be  easily  demonstrated  on  lactose 
litmus  gelatin  or  litmus  milk.  If  enough  acid  is  produced  the  lavender 
color  of  these  media  is  changed  to  red. 

Gas  Production. — Gas  production  is  controlled  by  inoculating 
sugar  bouillon  contained  in  a  fermentation  tube.  Various  kinds  of 
sugars,  such  as  saccharose,  dextrose,  maltose,  lactose,  mannite,  are 
used,  as  it  is  often  more  important  to  determine  the 
FIG.  71  varieties  of  sugar  which  are  or  are  not  split  up  by 

certain  species  of  bacteria.  The  closed  limb  of  the 
fermentation  tube  should  be  graduated  so  that  the 
amount  of  gas  formed  can  be  estimated  approxi- 
mately from  day  to  day  by  the  readings.  After  the 
formation  of  gas  has  ceased  the  proportion  of 
carbon  dioxid  and  hydrogen  present  can  be  ap- 
proximately estimated  by  the  following  method: 
Fill  the  open  bulb  of  the  tube  completely  with  a 
2  per  cent,  caustic  soda  solution.  Close  the  bulb 
with  the  thumb  or  a  rubber  stopper  and  invert 
the  tube  several  times  so  that  the  gas  is  intimately 
Fermentation  mixed  with  the  fluid,  which  now  contains  caustic 
tube.  soda.  The  latter  will  absorb  the  carbon  dioxid. 

Return  the  remainder  of  the  gas  to  the  closed  end 
of  the  tube,  remove  the  thumb  or  stopper  and  allow  the  fluid  to  cool 
so  that  the  residual  gas  is  no  longer  expanded  by  heat.  The  loss 
in  gas  represents  the  carbon  dioxid  and  the  balance  is  hydrogen. 
The  proportion  of  carbon  dioxid  to  hydrogen  is  often  characteristic 
for  certain  bacteria. 

Sulphuretted  Hydrogen. — To  ascertain  the  formation  of  sulphuretted 
hydrogen  a  special  culture  medium  known  as  iron  peptone  or  lead 
peptone  is  necessary.  These  culture  media  are  prepared  as  follows: 

1.  Take  peptone,  30  grams,  shake  with  water  heated  to  60°  C. 

2.  Wash  emulsion  into  a  liter  flask  with  80  c.c.  of  water. 

3.  Add  chloride  of  sodium,  5  grams,  and  phosphate  of  sodium,  3 
grams,  and  make  up  to  1000  c.c. 

4.  Heat  for  thirty  minutes  in  a  water  bath  or  steam  sterilizer  to 
dissolve  completely,  then  filter  clear. 

5.  Fill  into  tubes  10  c.c.  to  each  and  add  0.1  c.c.  of  a  2  per  cent, 
neutral  solution  of  tartrate  of  iron.    This  causes  a  yellowish-white 
precipitate  to  form. 

6.  Sterilize  for  twenty  minutes  on  three  consecutive  days. 

The  lead  peptone  is  prepared  in  the  same  manner  except  that  0.1 
c.c.  of  a  1  per  cent,  neutral  solution  of  lead  acetate  replaces  the  tar- 
trate of  iron  added  to  each  tube.  The  tubes  are  inoculated  with  the 


NITRITE  FORMATION  141 

bacterium  to  be  investigated  and  kept  in  the  incubator,  together  with 
non-inoculated  control  tubes.  If  hydrogen  sulphide  is  formed,  a 
brownish-black  to  black  precipitate  appears. 

Ammonia. — The  detection  of  ammonia  is  more  complicated.  It 
requires  distillation  after  the  culture  has  been  grown  in  several  hun- 
dred cubic  centimeters  of  the  culture  medium,  and  the  media,  of 
course,  must  be  prepared  with  ammonia-free  reagents,  including 
ammonia-free  water.  The  distillate  is  tested  with  Nessler's  reagent, 
which  gives  a  yellow  color  in  the  presence  of  free  ammonia.  The 
intensity  of  the  color  depends  upon  the  amount  of  ammonia  formed, 
and  the  test  can  be  arranged  as  a  colorimetric  quantitative  method. 

Indol. — Indol  is  not  infrequently  produced  in  the  growth  of  certain 
bacteria.  These  must  be  inoculated  into  Dunham's  peptone  water 
(see  above)  kept  in  tubes  which  have  been  incubated  for  several  days. 
Before  making  the  test  the  tubes  are  cooled  in  running  water  and  then 
the  following  reagents  are  added:  (1)  A  few  drops  of  a  1  to  10,000 
sodium  nitrite  watery  solution;  (2)  chemically  pure  sulphuric  acid, 
drop  by  drop,  until  about  1  c.c.  has  been  added  to  the  10  c.c.  con- 
tained in  the  culture  tube.  Red  discoloration  indicates  the  presence 
of  indol.  If  a  nitrite  has  been  formed  in  the  growth  the  red  color 
will  appear  on  the  addition  of  the  pure  strong  sulphuric  acid  alone. 

Phenol,  or  Carbolic  Acid. — Some  bacteria  also  produce  phenol,  or 
carbolic  acid.  To  ascertain  its  formation  about  100  c.c.  of  a  nutrient 
bouillon  are  inoculated.  After  growth  has  continued  for  several  days, 
approximately  one-fifth  to  one-sixth  of  the  fluid  is  distilled  over.  The 
distillate  contains  the  carbolic  acid.  It  is  tested  by  adding  first  a 
few  drops  of  lactic  acid  and  then  gradually  a  dilute  solution  of  the 
sesquichloride  of  iron.  If  phenol  is  present  an  amethyst-violet  color 
is  produced. 

Nitrite  Formation. — Some  bacteria  possess  the  power  to  reduce 
nitrates  to  nitrites.  This  characteristic  can  be  ascertained  by  inocu- 
lating the  nitrate  solution  described  in  the  preceding  chapter  with 
such  bacteria.  The  cultures  are  best  kept  for  one  week  at  a  temper- 
ature of  28°  C.  Two  solutions  are  necessary  for  the  nitrite  test: 

1.  Sulphanilic  acid  0.5  gram,  dissolved  in  150  c.c.  acetic  acid  of 
specific  gravity  1.04. 

2.  Amidonaphthalin  acetate  0.1  gram,  boiled  in  20  c.c.  distilled 
water,  then  filtered  through  cotton  and  diluted  with  180  c.c.  dilute 
acetic  acid. 

Before  use,  mix  equal  quantities  of  1  and  2,  and  add  2  c.c.  of  the 
mixture  to  3  c.c.  of  the  culture.  A  red  color  indicates  the  formation 
of  nitrites,  and  its  intensity  corresponds  to  the  smaller  or  greater 
amount  of  nitrites  present.  In  this,  as  in  other  similar  tests,  control 
tests  must  be  made  with  non-inoculated  tubes,  which  are  kept  in 
the  incubator  for  the  same  length  of  time. 


142     CULTURE  MEDIA  IN  RELATION  TO  METABOLIC  PRODUCTS 


QUESTIONS. 

1.  How  can  it  be  shown  whether  bacteria  in  their  growth  produce  a  proteo- 
lytic  ferment  or  not? 

2.  What  other  enzymes  are  produced  by  bacteria  in  their  growth? 

3.  What  is  an  enzyme? 

4.  Describe  the  preparation  of  culture  media  containing  starch. 

5.  Describe  the  preparation  of  a  bouillon  absolutely  free  from  sugar. 

6.  Why  is  the  agar  medium  not  adapted  for  preparing  a  sugar-free  culture 
soil? 

7.  What  is  diastase  ? 

8.  What  is  its  effect  upon  starch? 

9.  What  test  can  be  used  to  demonstrate  the  presence  of  amylodextrin,  ery- 
throdextrin,  and  achroodextrin?    What  are  the  color  reactions  of  this  test? 

10.  What  sugar  is  formed  by  the  action  of  diastase  upon  starch? 

11.  What  is  meant  by  an  amylolytic  ferment? 

12.  What  is  Fehling's  solution?    What  reaction  occurs  if  sugar  is  boiled  with 
it? 

13.  What  is  the  meaning  of  the  statement  that  sugar  is  a  reducing  substance  ? 

14.  Describe  the  determination  of  the  special  kind  of  sugar  present  in  a  solu- 
tion. 

15.  What  are  rennet  and  "lab"  enzymes  ?    How  can  they  be  detected  in  bacterial 
growth  ? 

16.  How  is  the  acid  production  of  bacteria  demonstrated  ?    How  is  the  amount 
of  acid  formed  determined  accurately? 

17.  How  can  alcohol  formation  be  detected? 

18.  What  apparatus  is  necessary  to  determine  the  formation  of  gas  in  bac- 
terial cultures? 

19.  Describe  the  test  to  determine  whether  part  of  the  gas  formed  is  carbon 
dioxide. 

20.  What  other  gas  is  usually  formed  with  carbon  dioxide  from  sugar? 

21.  What  culture  media  are  used  to  determine  whether  hydrogen  sulphide  is 
formed?    What  reaction  shows  the  formation  of  this  gas? 

22.  What  reagent  is  used  to  detect  the  formation  of  Ammonia? 

23.  What  is  indol?    What  culture  medium  is  used  to  demonstrate  its  forma- 
tion? 

24.  Describe  the  test  for  indol. 

25.  Describe  the  test  for  the  detection  of  carbolic  acid. 

26.  What  kind  of  a  process  is  the  change  of  nitrates  to  nitrites? 

27.  What  culture  medium  is  used  to  demonstrate  the  formation  of  nitrites? 

28.  Test  for  nitrites? 


CHAPTER    XII. 

METHODS   OF  OBTAINING  PURE   CULTURES  FROM   PATHOLOGIC 
MATERIAL  AND  OTHER  SOURCES. 

AFTER  the  bacteriologist  is  in  possession  of  various  types  of  sterile 
culture  media  he  may  undertake  the  preparation  of  pure  cultures 
from  pathologic  lesions  due  to  pathogenic  bacteria  or  from  other 
materials,  such  as  milk,  water,  food,  etc.  In  some  cases  this  is  a 
very  simple  and  easy  task.  For  example,  if  an  animal  has  a  deep- 
seated  abscess  which  has  not  broken  by  ulceration  and  the  bacterial 
cause  is  to  be  determined  the  procedure  would  be  as  follows:  Have 
in  readiness  a  number  of  culture  tubes,  some  slides,  a  platinum  loop, 
an  alcohol  lamp,  several  knives,  and  what  is  required  to  sterilize  the 
skin. 

1.  An  assistant  shaves  and  cleanses  the  skin  with  soap  and  water, 
strong  alcohol,  solution  of  corrosive  sublimate  1  to  500  to  1000.    The 
skin   is   then   dried   with  sterile  cotton  and  covered  with  the  same 
material. 

2.  The  knife  is  heated  over  an  alcohol  flame,  unless  it  is  kept  in  a 
small  portable  instrument  sterilizer.     After  the  removal  of  the  sterile 
cotton  an  incision  is  made  which  opens  up  the  abscess. 

3.  In  the  meantime  the  one  who  is  to  obtain  the  pure  culture  has 
prepared  himself  as  follows:     He  holds  two  culture  tubes  in  his  left 
hand.    Their  upper  ends  have  previously  been  heated  over  the  flame, 
which  destroyed  all  bacteria  that  may  have  collected  externally  on 
the  cotton.    When  everything  is  ready  the  cotton  plugs  are  removed 
by  a  rotary  twist  and  held  between  the  second  and  third  or  third  and 
fourth  fingers  of  the  left  hand.     The  tubes  must  be  held  obliquely, 
not  vertical,  because  in  this  position  bacteria  from  the  air  may  fall 
into  them,  nor  horizontally,  because  the  condensed  water  would  then 
run  out. 

4.  The  platinum  loop  is  heated  over  the  flame,  allowed  to  cool  for 
a  few  seconds,  and  dipped  into  the  pus  in  the  abscess  cavity  and 
then  introduced  into  the  first  tube.    There  it  is  dipped  into  the  con- 
densed water  and  rubbed  over  the  slanting  surface  of  the  culture 
medium.    This  procedure  is  called  inoculating  the  culture  tube. 

5.  After  the  first  tube  has  been  inoculated  the  platinum  loop   is 
again  sterilized  and  a  second  tube  is  inoculated  like  the  first  one. 

6.  The  platinum  loop  is  again  sterilized  and  laid  aside  and  the 
right  hand,  now  free,  closes  the  two  inoculated  tubes  with  the  cotton 
plugs;  these  are  again  burned  superficially  over  the  flame  (this  is 
called  flamed)  and  set  aside. 


144    PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

7.  The  platinum  loop  is  once  again  brought  into  use  to  make 
some  smears  on  the  slides  or  cover-glasses  which  have  been  in  readi- 
ness for  this  purpose.  When  working  around  animals  it  is  generally 
better  to  use  slides,  because  they  can  be  handled  more  easily  than 
the  fine  delicate  cover-slips,  and  the  subsequent  preparation  by  air 
drying,  fixing,  and  staining  is  the  same  for  either.  If  only  one  species 
of  bacterium  were  present  in  the  abscess  and  the  work  of  cleansing 
the  surface  and  opening  the  abscess  cavity  was  aseptically  performed, 
pure  cultures  should  be  obtained  in  the  two  test-tubes  inoculated. 

Plates  and  Petri  Dishes. — The  procedure  just  described  will  give 
results,  but  a  much  better  method  is  to  prepare  plates.     This  was 
first  practised  by  Robert  Koch,  but  his  method  was  somewhat  com- 
plicated and  easily  leads  to  contamination,  hence  it  has  been  largely 
replaced  by  the  use  of  the  Petri  dish.     The  latter 
FlG-  72  is  a  small,  circular  glass  vessel  about  four  to  six 

inches  in  diameter,  one-half  inch  high,  and  pro- 
vided with  a  cover.  These  Petri  dishes  must  be 
sterilized  before  use  in  the  hot-air  sterilizer,  and 
it  is  advantageous  when  they  have  to  be  taken  to 
Petri  dish.  a  distant  place  to  wrap  them  in  paper  which  has 

been  sterilized  with  them  and  which  is  only  ic- 
moved  just  before  they  are  to  be  used.  The  paper,  of  course,  is 
not  necessary  when  the  dishes,  after  cooling,  are  taken  from  the 
sterilizer  and  used  at  once  in  the  laboratory. 

Method  of  Pouring  Plates  or  Preparing  Petri  Dishes. — This 
method  must  always  be  used  when  there  is  any  chance  of  contami- 
nation of  the  material  to  be  examined,  and  is  at  all  times  better  than 
simply  inoculating  two  tubes,  as  previously  described.  The  method 
of  preparing  Petri  dishes  is  practised  as  follows: 

Take  four  agar  tubes  (if  gelatin  tubes  are  taken  the  procedure 
is  the  same,  except  as  to  the  temperature  figures  given)  and  melt 
the  contents  in  a  water  bath.  Open  one  of  the  tubes  and  introduce 
a  thermometer.  This  tube  is  not  to  be  used  for  inoculation  but 
merely  for  the  control  of  the  temperature.  After  the  agar  has  been 
melted  in  all  the  tubes,  allow  it  to  cool  down  to  about  50°  C.  Take 
the  tubes  out  of  the  water  bath.  Flame  the  upper  ends,  remove  the 
cotton  plugs,  and  place  the  three  tubes  in  a  slanting  position  on  the 
table.  This  can  be  done  with  the  aid  of  an  ordinary  small  slide  box, 
paper  box,  or  small  wire  rack.  Inoculate  tube  No.  1  with  platinum 
loop  (properly  sterilized)  three  times  from  the  pus.  Shake  tube  No.  1, 
always  keeping  it  in  a  slanting  position,  so  that  microorganisms  from 
the  air  cannot  fall  into  it.  Inoculate  tube  No.  2  three  times  with  the 
platinum  loop  (previously  sterilized)  from  tube  No.  1.  Shake  the 
contents  of  No.  2  well  and  finally  inoculate  No.  3  from  No.  2  in  the 
same  manner.  Shake  No.  3  well.  Now  pour  the  still  fluid  contents  of 
the  three  tubes  into  three  sterile  Petri  dishes  ready  for  the  purpose. 
This  pouring  of  the  Petri  dishes  is  done  as  follows:  Heat  the  upper 


PLATES  AND  PETRI  DISHES  145 

margin  of  the  culture  tube  over  a  flame.  Lift  up  the  lid  of  the  Petri 
dish  a  little,  but  be  careful  that  it  protects  the  dish  from  the  micro- 
organisms falling  into  it  from  the  air.  As  soon  as  the  fluid  is  trans- 
ferred from  the  culture  tube  cover  the  Petri  dish  again  with  its  lid. 
Do  this  with  all  three  tubes  and  allow  the  agar  in  the  dishes  to  cool 
as  rapidly  as  possible.  This  is  best  accomplished  by  cooling  them 
in  the  refrigerator  before  they  are  used.  When  the  agar  has  set  in 
the  Petri  dishes  these  can  be  labelled  as  Nos.  1,  2,  and  3,  corresponding 
to  the  tubes,  and  placed  in  the  incubator,  lids  down,  and  the  dish 
proper,  containing  the  agar  on  top.  This  is  done  so  that  the  con- 
densed water  which  forms  in  the  incubator  will  collect  in  the  lid  and 
not  on  the  agar,  where  it  would  do  harm. 

Object  of  Preparing  Petri  Dishes. — This  is  explained  by  the  follow- 
ing considerations :  Tube  No.  1  has  been  inoculated  with  pus  from  the 
abscess,  and  it  would  be  reasonable  to  assume  that  several  thousand 
cocci  which  are  the  cause  of  the  suppurative  abscess  and  a  few  hun- 
dred contaminating  bacteria  have  been  introduced.  The  pus  has  been 
diluted  in  the  agar  of  tube  No.  1,  and  when  No.  2  has  been  inoculated 
from  No.  1  there  will  be  perhaps  in  No.  2  several  hundred  cocci  and 
twenty  to  thirty  of  the  contaminating  organisms,  while  in  tube  No.  3, 
inoculated  from  the  very  dilute  pus  of  No.  2,  there  will  be  only  twenty 
cocci  and  one  or  two  of  the  contaminating  organisms.  As  soon  as 
the  agar  has  become  solid,  each  individual  bacterium,  or  perhaps  a 
small  group  of  individual  bacteria  which  have  clung  together  in  spite 
of  the  energetic  shaking  and  mixing,  is  fixed  in  a  definite  place,  and 
at  these  places  colonies  of  bacteria,  composed  of  many  millions, 
which  can  be  seen  either  with  the  naked  eye  or  with  a  hand  lens  or  low 
magnification  of  the  microscope,  have  developed  from  the  single  bacte- 
rium or  small  group  within  the  next  twenty-four  to  forty-eight  hours. 
Petri  dish  No.  1,  or,  as  it  is  often  called,  plate  No.  1,  will  develop  so 
many  colonies  in  the  incubator  within  the  next  twenty-four  hours 
that  they  soon  become  confluent  and  cannot  be  picked  up  individually. 
Plate  No.  3  is  generally  the  best  one.  It  contains  perhaps  ten  to  twenty 
colonies  of  the  coccus  and  one  or  two  colonies  of  the  contaminating 
microorganism.  The  two  different  types  can  often  be  distinguished 
by  the  naked  eye  from  the  appearance  of  their  colonies,  but  at  times 
it  may  be  necessary  to  make  microscopic  examinations  before  their 
character  can  be  recognized.  It  must  not  be  supposed  that  the  organ- 
ism present  in  twenty  colonies  is  always  the  cause  of  the  pathologic 
lesion,  and  that  the  one  represented  by  one  or  two  colonies  is  the 
contaminating  microbe.  Sometimes  this  is  not  the  case,  hence  it  is 
often  necessary  to  find  out  by  animal  experiments  which  is  the  causa- 
tive and  which  the  contaminating  bacterium.  On  the  other  hand, 
it  is  often  possible  to  decide  immediately  which  of  the  two  is  really 
the  cause  of  the  pathologic  lesion.  For  instance,  when  examining 
the  pus  of  an  abscess  as  described  above,  and  colonies  of  Staphylo- 
coccus  pyogenes  aureus  and  colonies  of  the  hay  bacillus  are  found,  it 
10 


146          PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

is  obvious,  of  course,  that  the  former  pathogenic  bacterium  is  respon- 
sible for  the  suppuration,  while  the  latter  harmless  saprophyte 
represents  an  accidental  contamination. 

When  there  are  on  one  or  more  of  the  Petri  dishes  discrete  (i.e.)  not 
confluent)  colonies  and  the  microscopic  examination  of  stained  cover- 
glass  preparations  shows  these  to  contain  one  microorganism  only 
which  is  considered  the  pathogenic  causative  bacterium  and  which 
is  desired  in  pure  culture,  another  set  of  Petri  dishes  should  be  pre- 
pared from  this  colony.  This  second  set,  provided  it  is  inoculated 
from  an  uncontaminated  colony,  should  contain  colonies  of  one  type 
only.  From  one  of  the  colonies  of  the  second  set  of  plates  a  number 
of  culture  tubes  of  various  media  should  then  be  inoculated  for  the 
further  study  and  identification  of  the  organism. 

"Fishing"  for  Colonies. — It  is  sometimes  not  easy  to  find  the  young 
small  colonies  which  may  have  developed  after  twenty-four  hours  in 
the  Petri  dishes,  and  it  may  not  only  be  necessary  to  hunt  for  them 
with  the  low  power  of  the  microscope,  but  also  to  remove  some  of 
the  material  of  such  a  colony  with  the  platinum  loop  while  looking 
through  the  instrument.  This  procedure  is  called  fishing  for  the 
colony.  It  is  not  an  easy  one  for  the  beginner,  and  in  order  to  facili- 
tate the  work  a  special  instrument  with  large  stage  and  low-power 
lenses  having  a  large  field  of  vision  is  used. 

Contamination  with  Moulds. — Plates  and  Petri  dishes  are  often, 
in  spite  of  all  precautions,  contaminated  with  moulds  which  have 
fallen  from  the  air  upon  the  culture  soil.  It  is,  as  a  rule,  quite  easy 
to  distinguish  the  mould  colonies  from  the  bacterial  colonies.  The 
former,  however,  may  grow  very  rapidly,  and  if  moulds  are  discovered, 
subcultures,  or  transplants,  should  be  made  at  once. 

Modifications. — Sometimes  it  is  very  difficult  to  pour  Petri  dishes 
on  account  of  the  surroundings,  as,  for  instance,  in  a  barn.  In  such 
places  a  simple  method  of  dilution  is  practised  which  often  gives  very 
good  results.  The  method  is  as  follows : 

Have  ready  a  number  of  slanting  agar  tubes.  They  may  be 
kept  upright  in  the  vest  or  coat  pocket  if  there  is  no  chance  to  place 
them  on  a  table.  Sterilize  the  platinum  loop,  flame  tube  No.  1  over 
an  alcohol  lamp  held  by  an  assistant,  open  tube  and  inoculate  the 
condensed  water  of  the  agar  three  times  with  the  platinum  loop. 
Sterilize  the  latter,  open  tube  No  1  again,  enter  with  a  sterile  loop 
and  rub  the  condensed  water  well  over  the  agar  surface.  Now  inoculate 
tube  No.  2  from  No.  1.  Sterilize  loop  again  and  enter  tube  No.  2  and 
rub  over  surface  of  agar  and  inoculate  with  the  material  obtained  from 
the  condensed  water  of  tube  No.  3.  In  this  manner  a  dilution  of  the 
original  material  is  obtained  which  will  lead  to  the  formation  of  a  few 
discrete  individual  colonies  in  tube  No.  3  or  No.  4.  If  the  tubes  are 
dry  and  do  not  contain  any  condensed  water,  then  the  same  method 
may  be  practised  by  simply  rubbing  the  loop  each  time  over  the 
whole  surface  of  the  agar,  sterilizing  the  platinum  each  time  between 


PREPARATIONS  FROM  THE  CIRCULATING  BLOOD         147 

the  inoculations  of  the  different  tubes.  In  this  manner  the  desired 
dilution  is  obtained,  and  results  in  the  formation  of  non-confluent 
individual  colonies. 

Another  modification  of  the  original  plate  method  of  Koch  has  been 
devised  by  Esmarch;  its  details  are  the  following:  Tubes  with  fluid, 
gelatin  or  agar,  cooled  down  to  near  the  point  of  solidification  of  the 
medium,  are  inoculated  successively  as  described  above,  but  their 
contents  are  not  poured  out.  Instead  the  tubes  are  rolled  on  an  ice 
block  until  the  medium  has  formed  a  solid  coating  on  the  inside  of 
the  test-tube.  The  developing  colonies  can,  as  in  the  case  of  the  plates 
or  Petri  dishes,  be  examined  under  the  microscope. 

FIG.  73  FIG.  74 


Method  of  holding  tubes  during  inoculation.  Esmarch's  magnifier  for  counting  colo- 

(McFarland.)  nies     of     bacteria    in     Esmarch     tubes. 

(McFarland.)    •' 

Impression  Preparation. — When  a  plate  or  Petri  dish  has  developed 
colonies  it  is  often  desirable  to  study  the  finer  details  of  the  arrange- 
ment of  the  bacteria  forming  the  colony.  This  can  be  done  by  placing 
a  clean  dry  cover-glass  over  the  colony  and  pressing  it  down  tolerably 
firmly  on  the  culture  soil.  In  this  manner  an  impression  of  the  colony 
on  the  cover-glass  is  obtained.  The  latter  is  then  lifted  with  a  pair  of 
fine  forceps,  air  dried,  fixed,  stained  in  the  usual  manner,  and  exam- 
ined with  the  oil-immersion  lens.  The  microscopic  specimen  obtained 
in  this  manner  is  known  as  an  impression  preparation  or  "Klatsch- 
Prseparat." 

Preparations  from  the  Circulating  Blood. — It  is  often  desirable  or 
necessary  to  make  pure  cultures  from  bacteria  which  may  be  present 
in  the  circulating  blood  of  a  sick  animal  or  person.  This  is  done  in 
the  following  manner:  The  animal  is  first  restrained.  Next  shave 
and  cleanse  the  skin  so  that  it  is  sterile.  Have  on  hand  a  sterile 
(preferably  all  glass)  syringe  and  a  number  of  flasks  containing  from 


148    PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

50  to  100  c.c.  of  a  fluid  culture  medium.  When  everything  is  ready, 
the  syringe  is  taken  out  of  the  small  instrument  sterilizer  or  out  of  its 
receptacle  in  which  it  has  been  sterilized.  The  point  of  the  needle 
is  rapidly  flamed  and  the  needle  is  plunged  into  the  animal's  vein, 
which  has  been  previously  compressed  centrally  just  beyond  the  point 
where  the  puncture  is  to  be  made.  When  the  barrel  of  the  syringe  is 
full  of  blood  and  the  needle  has  been  withdrawn,  the  culture  flasks 
are  opened  by  an  assistant  and  the  blood  transferred  to  them  in  the 
proportion  of  1  to  5  c.c.  to  50  to  100  c.c.  of  the  fluid  culture  medium. 
The  flasks  are  closed  immediately,  the  cotton  plugs  flamed  and  the 
receptacles  placed  in  the  incubator.  After  twenty-four  hours  some  of 
the  contents  of  the  flasks  are  poured  into  centrifuge  tubes  (carefully, 
so  as  not  to  permit  any  contamination  from  the  air).  The  tubes  are 
next  centrifuged  and  cover-glasses,  which  are  stained  and  examined 
in  the  usual  manner,  are  prepared  from  the  sediment.  If  any  organ- 
isms are  found,  culture  tubes  with  solid  media  are  inoculated  from  the 
fluid  media.  It  is  also  advisable  to  pour  plates  (Petri  dishes)  to 
detect  contaminations  if  any  are  present. 

Inoculation  from  Postmortem  Material. — It  often  becomes  necessary 
to  obtain  pure  cultures  from  postmortem  material.  If  the  animal 
has  not  been  dead  long,  or  if  immediately  after  death  the  body  has 
been  placed  on  ice,  the  procedure  is  not  at  all  difficult,  and  is  as  follows : 
If  the  animal  is  a  small  one,  not  larger  than  a  medium-sized  dog,  it 
is  best  to  place  the  cadaver  on  a  board  and  stretch  out  the  four  legs 
by  fastening  them  with  twine  to  four  nails  or  blocks  driven  into  the 
four  corners  of  the  board.  An  incision  from  the  sternum  to  the  sym- 
physis  pubis  is  made  with  a  sterile  knife  and  the  skin  is  loosened  from 
the  thoracic  and  abdominal  walls  and  tacked  to  the  board.  The  peri- 
toneum is  then  incised  and  the  bony  thoracic  wall  is  removed  by  cutting 
through  the  costal  cartilages.  This  is  all  done  with  sterile  instruments 
which  have  been  changed  several  times.  When  the  heart  is  exposed 
it  is  raised  with  a  pair  of  sterile  forceps  and  the  wall  of  the  right 
ventricle  is  cut  with  sterile  scissors  or  knife.  As  soon  as  the  blood 
flows  out,  culture  tubes  are  inoculated  with  it,  using  the  platinum  loop. 
Cultures  may  also  be  inoculated  from  the  other  organs  with  the  aid 
of  a  very  strong  platinum  loop.  This  is  heated,  and  while  still  hot  is 
pushed  into  the  parenchyma  of  the  organ,  where  it  is  left  a  few  seconds 
until  cool  and  then  pushed  in  a  little  farther  and  small  bits  of  tissue 
are  withdrawn  from  the  organ.  With  the  particles  so  obtained  culture 
media  are  inoculated.  The  procedure  differs  somewhat  with  large 
animals.  The  heart's  blood  is  best  obtained  in  the  same  manner, 
but  the  other  organs  should  be  removed  and  placed  upon  a  table. 
A  large  flat  knife  is  then  heated  over  a  flame  and  is  pressed,  while 
quite  hot,  on  the  organ.  This  singes  the  surface.  The  spot  so  treated 
is  next  cut  into  with  a  sterile  knife  and  a  platinum  loop  introduced 
into  the  incision  to  obtain  some  juice  for  inoculating  culture  tubes  or 
pouring  plates. 


STAB  CULTURES  149 

Subcultures,  or  Transplants. — After  bacteria  have  been  obtained  in 
pure  cultures  by  one  of  the  methods  described,  it  is  always  necessary 
to  prepare,  from  time  to  time,  fresh  cultures,  because  numerous  micro- 
organisms soon  show  a  tendency  to  die  out  in  an  artificial  culture 
medium,  on  account  of  the  accumulation  of  their  own  metabolic 
products  or  for  other  reasons.  The  transfer  of  an  older  culture  to  a 
new,  fresh,  sterile  culture  medium  is  made  in  such  a  manner  that 
the  danger  of  air  and  other  contaminations  is  reduced  to  a  minimum. 
This  is  accomplished  by  flaming  the  cotton  and  upper  ends  of  the 
tubes  or  flasks  containing  the  growth,  as  well  as  those  of  the  new 
ones  to  be  inoculated;  or,  when  Petri  dishes  are  used,  by  only  lifting 
the  lid  sufficiently  to  allow  the  introduction  of  the  platinum  loop. 
The  procedure  of  inoculating  new  tubes  or  flasks  from  preexisting 
pure  cultures  is  called  making  a  subculture,  or  transplant  (trans- 
planting the  cultures). 

The  first  pure  culture  obtained  from  pathologic  or  other  material 
is  spoken  of  as  the  first  generation;  the  first  transplant  as  the  second, 
the  next  transplant  as  the  third  generation,  and  so  forth.  A  culture 
tube  should  always  be  so  labelled  that  it  clearly  shows  the  kind  of 
culture  medium,  the  kind  of  microorganism,  its  derivation,  the  gener- 
ation, the  date  wThen  the  original  inoculation,  or  transplant,  was  made, 
and  something  about  the  manner  in  which  it  has  been  raised.  A 
sufficiently  designated  culture  would,  for  example,  show  the  following 
inscription  on  the  label  • 

Glycerin  Agar 
Bacillus  Anthracis 
Blood  of  mouse  dead  from  Anthrax 
Fourth  Generation 
January  15,    1910 
(Incubator,  kept    aerobically) 

It  is,  of  course,  not  necessary  to  label  as  elaborately  the  cultures  used 
by  students  for  laboratory  exercises,  but  the  culture  medium,  the 
species  of  bacterium,  and  the  date  of  inoculation  must  always  be  given, 
and  they  should  be  repeated  on  the  label  of  the  microscopic  prepar- 
ation. It  is  also  desirable  to  indicate  on  the  latter  the  stain  used. 

Streak  Cultures. — The  described  method  of  making  an  original 
culture  or  a  transplant  by  drawing  the  platinum  loop  over  the  slanting 
surface  of  agar  or  over  a  potato  half-cylinder  in  a  test-tube  is  called 
a  streak  culture.  There  is  no  special  name  for  the  inoculation  of  the 
fluid  media. 

Stab  Cultures. — Gelatin  of  any  kind  and  sugar  agar  are  generally 
not  prepared  in  test-tubes  in  a  slanting  position,  but  as  a  solid  cylinder 
at  the  bottom  of  the  tube.  This  condition  is  brought  about  by  keeping 
the  tubes  in  an  upright  position  at  the  time  when  the  media  solidify. 
Such  media  are  inoculated  with  the  platinum  wire  straightened  out 
into  a  needle.  The  outer  free  end  of  the  wire,  after  sterilization 
over  a  flame  and  subsequent  cooling,  is  brought  in  contact  with  the 


150         PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

bacterial  growth  and  the  needle  is  then  plunged  down  vertically  into 
the  solid  culture  medium.  This  method  of  inoculating  a  tube  is  called 
a  stick  or  stab  culture.  It  is  used  when  a  gelatin  tube  is  inoculated 
in  order  to  show  whether  liquefaction  takes  place  or  not,  and  it  is 
also  employed  in  the  preparation  of  anaerobic  cultures,  because  the 
deeply  inoculated  bacteria  are  to  a  great  extent  removed  from  the 
air  in  the  upper  part  of  the  tube. 

Shake  Cultures. — It  is  sometimes  desirable  to  distribute  the  inoculated 
material  evenly  in  a  solid  culture  medium.  The  latter  is  then  lique- 
fied and  cooled  down  to  near  its  point  of  solidification.  The  inocu- 
lation is  then  made  and  the  still  fluid  medium,  after  the  closure  of 
the  tube  with  the  cotton  plug,  is  shaken  violently  and  finally  allowed 
to  solidify  in  an  upright  position.  Such  a  shake  culture  will  readily 
permit  the  formation  of  gas  bubbles  in  the  developing  growth. 

Pure  Cultures  by  Preliminary  Animal  Inoculations. — It  is  sometimes 
impossible  to  obtain  the  causative  pathogenic  bacteria  in  pure  culture 
from  a  pathologic  discharge,  excretion,  or  tissue  by  direct  inoculation 
of  culture  media.  Even  the  plate  (Petri  dish)  method  is  not  available 
in  some  cases.  The  cause  of  tuberculosis,  the  tubercle  bacillus,  for 
instance,  grows  very  slowly,  and  in  tubercular  discharge  is  generally 
associated  with  other  bacteria.  If  tubes  were  inoculated  and  plates 
poured  from  a  material  of  this  kind  all  other  bacteria  present  would 
develop  days  before  the  tubercle  bacillus  had  a  chance  to  form  a 
colony.  In  a  case  of  this  kind  it  is  necessary  to  inoculate  the  material 
into  an  animal  susceptible  to  tuberculosis.  The  tubercle  bacillus  will 
multiply  in  its  body  and  be  present  in  certain  locations  and  structures 
in  pure  culture.  At  an  appropriate  time  the  animal  is  killed  and  under 
aseptic  precautions  some  of  the  tubercular  material  is  obtained  and 
brought  into  the  proper  culture  media  to  give  the  tubercle  bacillus  a 
chance  to  develop  and  form  a  pure  culture.  The  same  method  is 
generally  necessary  to  obtain  a  pure  culture  of  glanders  bacilli  from 
a  horse  suffering  from  glanders,  because  here  also  the  glanderous 
discharges  are  contaminated  by  many  bacteria  growing  more  rapidly 
than  the  bacillus  mallei.  Further  details  about  bacteria  which  have 
to  be  obtained  in  pure  cultures  in  this  indirect  manner  will  be  given 
in  the  chapters  devoted  to  such  particular  microorganisms. 

Anaerobic  Cultures. — Anaerobic  bacteria  which  do  not  develop  in  the 
presence  of  the  oxygen  of  the  atmospheric  air  have  to  be  cultivated 
under  special  arrangements  which  will  exclude  this  gas.  Various 
methods  have  been  developed  to  accomplish  this  end.  The  simplest 
method,  devised  by  R.  Koch,  consists  in  placing  a  sterile  piece  of 
mica  over  the  surface  of  a  plate  inoculated  with  an  anaerobic  germ. 
This  method  was  later  modified  by  replacing  the  mica  by  a  sterile 
glass  plate  and  sealing  the  latter  around  its  margins  by  sterile  gelatin 
or  agar  to  which  a  small  amount  of  some  antiseptic  had  been  added. 
Methods  of  this  type  have  been  largely  abandoned,  and  have  been 
replacedH)y*betterJdevices. 


REPLACING  THE  AIR  BY  A  HYDROGEN  ATMOSPHERE     151 

The  Stick  Culture  Method. — Anaerobic  cultures  may  be  raised  in 
stick  cultures.  For  this  purpose  it  is  well  to  have  a  somewhat  longer 
tube  containing  instead  of  10  c.c.,  15  to  20  c.c.  of  the  culture  medium. 
To  the  latter,  when  used  to  raise  anaerobic  stick  cultures,  a  reducing 
substance,  such  as  sugar,  or,  still  better,  formate  of  sodium,  is  often 
added.  Anaerobic  stick  cultures  can  also  be  made  as  follows :  A  culture 
tube  containing  the  usual  amount  of  medium  is  inoculated  as  a  stick 
culture.  The  medium  in  a  second  tube  is  melted  and  allowed  to 
cool  to  near  its  point  of  solidification.  Before  this  is  reached,  however, 
the  cotton  plug  and  the  upper  end  of  the  tube  are  flamed,  the  former 
removed  and  the  still  fluid  contents  of  the  tube  poured  into  the  stick 
culture  in  the  first  tube.  The  added  medium  will  successfully  exclude 
the  air  from  the  lower  inoculated  strata,  giving  the  anaerobic  germ  a 
chance  to  grow. 

Exclusion  of  Air  from  Fluid  Media. — A  simple  and  often  successful 
method  of  raising  anaerobic  germs  in  fluid  media  (bouillon,  milk,  etc.) 
is  the  following:  The  media,  shortly  before  use,  are  subjected  to  a 
prolonged  boiling,  which  drives  out  the  atmospheric  air.  The  culture 
flasks  without  being  in  any  way  disturbed  or  agitated,  which  would 
again  mix  the  culture  medium  with  atmospheric  air,  are  allowed  to 
cool,  and  are  then  at  once  inoculated.  In  the  meantime  there  should 
be  prepared  some  oil,  vaselin  or  paraffin  of  a  low  melting  point,  which 
has  been  sterilized  by  heat  and  allowed  to  cool.  As  soon  as  the  culture 
flasks  have  been  inoculated  some  of  the  oil,  vaselin  or  the  like  is 
poured  into  them.  These  substances,  lighter  than  water,  will  float  on 
the  surface  and  exclude  the  culture  medium  and  the  anaerobic  bacteria 
contained  therein  from  contact  with  the  atmospheric  air. 

Replacing  the  Air  by  a  Hydrogen  Atmosphere. — Anaerobic  bacteria 
can  be  raised  both  in  an  atmosphere  of  hydrogen  and  in  one  composed 
of  nitrogen  from  which  all  oxygen  has  been  removed.  When  the 
ordinary  air  is  to  be  replaced  by  hydrogen  it  is  necessary  to  use  an 
apparatus  developing  a  continuous  current  of  this  gas.  The  simplest 
and  safest  device  of  this  kind  is  a  Kipp  gas  generator.  It  consists  of 
two  glass  globes  (A  and  B)  joined  together  by  a  narrow  neck  and 
resting  on  a  base.  The  upper  globe  (^4)  possesses  a  lateral  tubular 
outlet  ( T)  closed  with  a  perforated  rubber  stopper  which  is  provided 
with  a  glass  tube  and  a  stopcock  (ST).  A  third  globe  (C)  is  generally 
shaped  like  a  separatory  funnel  with  a  narrow  conical  glass  tube 
fitting  air-tight  into  the  neck  (N)  of  the  upper  globe  (^4)  without 
completely  closing  the  passage  between  the  two  jointed  globes  at  (M ). 
When  this  apparatus  is  to  be  used  for  the  generation  of  hydrogen, 
the  rubber  stopper  with  the  gascock  at  ( T)  is  removed  and  pieces  of 
broken  glass  are  introduced  in  such  a  manner  that  they  collect  around 
the  long  glass  tube  in  A.  Next  granulated  zinc  is  introduced  into 
globe  A  in  the  same  manner.  The  zinc,  provided  that  the  broken 
glass  has  been  arranged  properly,  cannot  fall  into  B.  The  next  step 
is  to  open  the  stop-cock  at  ST  and  fill  B  through  the  upper  globular 


152 


PURE  CULTURES,  FROM  PATHOLOGIC  MATERIAL 


funnel  with  enough  sulphuric  acid,  considerably  diluted  (1  part 
H2SO4  to  9H2O)1  until  the  fluid  about  reaches  the  constriction 
between  the  two  united  glass  globes  (at  M).  The  stopcock  is  then 
closed  and  more  dilute  sulphuric  acid  is  poured  into  the  funnel  (C), 
where  it  remains  as  long  as  the  cock  is  closed.  The  tube  with  the 
stopcock  is  now  connected  with  several  Woulfe's  wash  bottles.  This 
is  done  to  remove  impurities  from  the  hydrogen  gas.  When  the  latter 
is  to  be  generated  the  stopcock  is  opened,  allowing  dilute  sulphuric 
acid  to  run  from  the  upper  funnel  into  the  globe  which  contains  the 
zinc.  As  soon  as  this  takes  place  the  development  of  hydrogen  begins 


FIG.  75 


Kipp  apparatus  and  accessories  for  generating  and  purifying  hydrogen  gas. 

and  the  gas  escapes  at  the  open  stopcock  into  the  wash  bottles,  and 
from  there  into  the  tubes  or  jars  which  contain  the  media  inoculated 
with  anaerobic  cultures.  The  flow  of  the  gas  can  be  regulated  at  the 
stopcock,  which  may  be  kept  wide  open  or  partially  closed  so  that 
only  a  moderate  amount  of  gas  escapes.  When  no  more  gas  is  needed, 
all  that  is  necessary  is  to  close  the  stopcock  at  st.  No  more  gas  can 
escape,  hence  the  hydrogen  accumulates  in  A,  and  presses  upon  the 

1  When  mixing  H2O  and  HjSO^  the  sulphuric  acid  has  to  be  poured  slowly  into  the  water. 
It  is  not  permissible  to  pour  water  into  strong  H2SO4,  because  the  latter  may  become  so  hot 
that  dangerous  consequences  may  be  brought  about.  The  dilute  sulphuric  acid  must  first 
have  become  cool  before  it  can  be  used  in  the  Kipp  apparatus. 


REPLACING  THE  AIR  BY  A  HYDROGEN  ATMOSPHERE     153 


Fia.  77 


level  of  the  fluid  in  A  and  displaces  it  downward.  As  soon  as  the  fluid 
has  receded  from  the  zinc  no  more  hydrogen  is  developed  and  an 
equilibrium  of  pressure  is  established  in  the 
apparatus.  Whenever  more  hydrogen  is 
needed  all  that  is  necessary  is  to  open  the 
stopcock,  when  the  fluid  from  the  funnel 
falls  into  the  lower  globe,  rises  into  the 
upper  globe,  comes  in  contact  with  the 
zinc,  and  hydrogen  is  at  once  developed. 
When  the  zinc  has  all  been  dissolved,  or 
when  the  dilute  acid  has  become  exhausted, 
it  is  necessary  to  refill  the  apparatus.  When 
working  with  hydrogen  it  is  necessary  to 
remember  that  the  gas  is  not  only  combus- 
tible, but  forms,  when  mixed  with  the  oxygen 
of  the  atmospheric  air  in  the  proportion  of 
two  volumes  to  one  volume  of  oxygen,  a  very 
explosive  gas.  Hydrogen  generated  in  the 
Kipp  apparatus  from  ordinary  commercial 
zinc  contains,  as  contaminations,  sulphur, 
arsenic,  and  also  some  oxygen.  These 
bodies  must  all  be  removed  before  the  gas 
can  be  used  in  the  anaerobic  cultures.  This 
is  accomplished  by  leading  the  impure 
oxygen  through  three  wash  bottles  contain- 
ing, respectively,  the  following  solutions: 
No.  1,  a  10  per  cent,  watery  solution  of 
nitrate  of  lead;  No.  2,  a  10  per  cent,  solu- 
tion of  nitrate  of  silver,  and  No.  3,  an  alkaline 
solution  of  pyrogallic  acid.  If  chemically 
pure  zinc  and  chemically  pure  sulphuric 
acid  diluted  with  distilled  water  is  used  then 


FIG.  76 


Novy  jar  for  anaerobic  cultures. 


Buchner's  anaerobic  tube. 
The  fluid  consists  of  pyro- 
gallic acid  dissolved  in  10 
per  cent,  soda  solution.  By 
Wilson's  method  the  tubes  are 
charged  with  pieces  of  caustic 
potash  covered  with  pyrogallic 
acid.  (Park.) 


154    PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

the  purification  of  the  hydrogen  is  much  simpler.  The  first  wash 
bottle  may  then  contain  a  solution  of  iodine  and  iodide  of  potash  and 
the  second  one  concentrated  H2SO4.  These  bottles  will  wash  and 
then  dry  the  gas. 

In  order  to  replace  the  atmospheric  air  in  the  tubes,  flasks,  or  Petri 
dishes  containing  the  anaerobic  cultures  by  hydrogen,  special  arrange- 
ments are  always  necessary  to  lead  the  gas  in  and  then  to  close  the 
culture  medium  container  in  an  air-tight  manner.  The  Novy  jar  is 
the  apparatus  easiest  to  handle.  It  comes  in  a  high  pattern  adapted 
for  tubes  and  flasks  and  in  a  low  pattern  adapted  for  Petri  dishes. 
This  jar  has  an  inlet  and  an  outlet  tube  which  can  be  closed  by  air- 
tight glass  stopcocks.  After  a  Novy  jar,  containing  culture  tubes  or 
plates,  has  been  filled  with  hydrogen,  it  is  well  to  seal  the  lid  and  the 
stopcocks  with  melted  paraffin  as  an  additional  precaution.  In 
order  to  see  whether  all  atmospheric  air  has  been  displaced  from  the 
Novy  jar  or  other  apparatus  used  the  following  test  should  be  made 
from  time  to  time.  The  outlet  tube  of  the  jar  is  connected  with  a 
small  rubber  tube  which  carries  at  its  outer  end  a  small  glass  tube 
bent  at  right  angles.  The  free  limb  of  this  rectangular  glass  tube 
is  lead  into  a  test-tube  held  with  its  closed  end  upward.  After  a  short 
time  the  glass  tube  is  withdrawn;  the  mouth  of  the  test-tube  rapidly 
closed  with  the  thumb  and  the  tube  now  inverted  so  that  its  mouth 
points  upward.  A  match  is  lit  and  held  at  the  mouth  of  the  test-tube, 
and  if  the  gas  is  pure  it  burns  with  a  blue,  non-luminous  flame;  if 
still  mixed  with  atmospheric  air  there  will  be  a  slight  explosion. 
This  test,  which  should  be  repeated  until  the  result  indicates  pure 
oxygen,  must  be  made  at  some  distance  from  the  Kipp  apparatus. 
The  latter  is  best  rigged  up  under  a  hood.  Where  none  is  present  the 
escaping  hydrogen  can  be  let  out  of  the  room  by  the  following  simple 
arrangement:  A  small  hole  is  made  with  an  auger  in  a  window  frame 
and  a  glass  tube  passed  through  the  hole.  This  glass  tube  is  con- 
nected with  the  outlet  tube  of  the  Novy  jar  by  a  piece  of  small  caliber 
rubber  tubing  and  the  hydrogen  gas  flowing  through  the  apparatus 
is  carried  out  of  doors. 

Removing  the  Oxygen  from  the  Air  by  Chemical  Means. — This 
method,  first  used  by  Buchner  for  the  cultivation  of  anaerobic  bacteria, 
is  based  upon  the  principle  of  absorbing  the  oxygen  of  the  air  in  a 
closed  vessel  by  an  alkaline  solution  of  pyrogallic  acid.  Applied  to 
single-culture  tubes  the  method  is  practised  as  follows :  A  large  test- 
tube,  into  which  the  much  smaller  culture  tube  fits  easily,  is  provided 
with  a  tightly  fitting  rubber  stopper.  One  gram  of  pyrogallic  acid 
and  10  c.c.  of  a  10  per  cent,  solution  of  potassium  hydrate  are  placed 
in  the  large  tube.  The  inoculated  culture  tube,  with  a  piece  of  thin 
string  fastened  around  the  mouth,  is  suspended  in  the  large  tube  and 
the  string  is  held  in  place  by  the  rubber  stopper  of  the  large  tube. 
The  latter  is  then  sealed  by  pouring  paraffin  on  top  of  the  rubber 
stopper  and  around  it.  After  this  the  tube  can  be  incubated.  If  a 


WRIGHT'S  METHOD  FOR  ANAEROBIC  CULTURES  155 

number  of  tubes  or  Petri  dishes  inoculated  with  anaerobic  germs  are 
to  be  treated  by  the  pyrogallic-acid  method,  anatomical  jars  may  be 
used  for  the  pupose.  Tubes  can  be  placed  in  a  slanting  position 
against  the  wall  of  the  jar,  but  when  Petri  dishes  are  used  some  device 
for  them  to  rest  upon  should  be  placed  in  the  bottom.  The  chemicals 
are  placed  in  the  jar,  its  lid  screwed  down  and  sealed  with  paraffin, 
and  the  jar  is  then  ready  to  go  into  the  incubator.  A  more  elaborate 
glass  jar  is  one  made  with  shelves  on  which  the  plates  containing  the 
cultures  may  rest.  This  can  be  used  with  either  the  hydrogen  or  the 
pyrogallic-acid  absorption  method. 

Anaerobic  Cultures  in  Hen's  Eggs. — Anaerobic  cultures  may  also  be 
raised  in  hen's  eggs.  The  latter  should  be  fresh  and  the  shell  must 
be  cleansed  externally  by  washing  in  a  bichlorid  solution  and  sub- 
sequently in  sterile  water,  and  finally  drying  with  sterile  cotton.  A 
suitable  spot  is  then  perforated  with  a  sterile  needle  and  the  inocu- 
lation is  made  with  a  slender  platinum  needle.  The  small  hole  is 
then  closed  with  hot  sealing  wax  and  the  whole  outer  surface  coated 
with  a  varnish.  Eggs  so  prepared  are  then  incubated  and  at  the 
proper  time  broken  and  their  contents  discharged  into  a  sterile  glass 
receptacle  for  microscopic  examination. 

The  preparation  of  anaerobic  cultures  by  the  removal  of  the  air 
from  the  container  of  the  culture  medium  by  an  air  pump  is  not  often 
practised  nowadays,  since  other  anaerobic  methods  are  much  simpler 
and  more  preferable. 

Wright's  Method  for  Anaerobic  Cultures. — Wright  has  devised  two 
methods  of  developing  anaerobic  cultures:  one  of  them  is  a  modifi- 
cation of  the  pyrogallic-acid  method  of  Buchner,  the  other  consists 
in  removing  all  air  from  the  fluid  culture  medium  by  a  special  arrange- 
ment of  the  glass  tube  which  contains  the  medium.  These  methods 
are  described  in  Mallory  and  Wright's  Manual  of  Pathological 
Technique.  The  first  method  is  applicable  to  cultures  in  test-tubes 
and  flasks;  the  details  are  as  follows: 

After  the  culture  medium  in  the  test-tube  has  been  inoculated  the 
cotton  stopper  is  pushed  into  the  test-tube,  so  that  the  top  is  about 
1.5  cm.  below  the  mouth  of  the  test-tube.  It  is  usually  desirable  to 
cut  off  a  part  of  the  protruding  portion  of  the  cotton  before  doing 
this.  This  cotton  of  the  stopper  should  be  of  a  kind  that  will  readily 
absorb  fluids.  Now  fill  the  space  in  the  tube  above  the  cotton  stopper 
with  dry  pyrogallic  acid  and  quickly  add  enough  of  a  strong  watery 
solution  of  sodium  hydrate  to  dissolve  it  all.  Avoid  pouring  on  an 
excess;  for  a  test-tube  f  inch  in  diameter  about  2  c.c.  will  be  ample. 
Then,  as  quickly  as  possible,  insert  a  rubber  stopper  firmly  in  the 
mouth  of  the  tube  so  as  to  close  it  tightly.  The  culture  is  then  ready 
to  be  set  aside  for  development.  The  solution  of  sodium  hydrate 
used  consists  of  one  part  of  the  former  dissolved  in  two  parts  of 
water.  If  done  properly  there  is  no  danger  of  contaminating  the 
culture  medium  from  the  alkaline  pyrogallic-acid  solution.  The 


156 


PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 


FIG.  78 


method  gives  good  results  in  obtaining  pure  cultures  of  the  tetanus 
bacillus. 

The  other  method  of  Wright's  is  as  follows:  The  apparatus 
consists  of  a  simple  arrangement  of  glass  and  rubber  tubes  enclosed 
in  an  ordinary  test-tube  with  a  plug  or  cotton  inserted  in  its  mouth, 
as  in  an  ordinary  culture  tube.  The  construction  of  the  apparatus 
is  shown  in  Fig.  78.  A  is  a  glass  tube,  somewhat  constricted  at  each 

extremity.  B  and  C  are  short  pieces  of  small 
rubber  tubing.  D  is  a  glass  tube,  in  the 
upper  extremity  of  which  a  small  plug  of 
cotton  is  inserted;  E  is  a  piece  of  rubber 
tubing.  The  test-tube  contains  a  quantity 
of  the  fluid  culture  medium.  When  it  is 
desired  to  make  an  anaerobic  culture  the 
fluid  in  the  test-tube  is  inoculated  in  the 
usual  way.  The  fluid  is  then  sucked  up  into 
the  system  of  glass  and  rubber  tubes  to  a 
level  above  the  rubber  tube  C.  WThen  it 
has  reached  this  level  the  rubber  tube  E  is 
compressed  between  the  fingers  to  prevent 
the  down  flow  of  the  fluid,  and  the  system 
of  tubes  is  then  pushed  downward  in  such  a 
way  as  to  bend  the  rubber  tubes  B  and  C  as 
shown  in  Fig.  78.  If  the  test-tube  and  the 
inner-tube  system  are  of  suitable  size  the 
rubber  tubes  mentioned  will  remain  in  this 
bent  position.  The  fluid  in  the  tube  A  is 
thus  contained  in  a  water-tight  space,  because 
the  rubber  tubes  B  and  C,  when  bent  to  the 
angle  shown  in  Fig.  78,  are  closed  water- 
tight. Cover-glass  preparations  may  be 
made  from  the  culture  fluid  by  straight- 
ening out  the  system  of  tubes  and  allowing 
the  fluid  in  them  to  flow  into  the  test-tube, 
where  it  is  accessible  to  the  platinum  loop 
in  the  usual  way.  In  using  this  method 
Wright's  method  of  making  ft  •  f  course  necessary  that  most  of  the 

anaerobic     cultures     in       fluid       .      .'  .  J 

media.    (Maiiory  and  Wright.)  air  in  the  culture  fluid  be  expelled  betorc  it 

is  inoculated.     This  is  easily  done  by  boiling 

the  culture  fluid  over  the  flame  of  a  Bunsen  burner  without  removing 
the  inner  system  of  tubes,  and  then  cooling  the  apparatus  by  placing 
it  in  cold  water. 

Incubators  or  Thermostats. — Most  pathogenic  bacteria  grow  best  at 
the  temperature  of  the  body  of  susceptible  animals,  and  it  is,  therefore, 
necessary  to  raise  artificial  cultures  in  the  incubator,  thermostat,  or 
brood  oven  at  a  temperature  of  about  37°  to  38°  C.  Modern  incu- 
bators for  bacteriologic  work  are  generally  constructed  of  copper, 


INCUBATORS  OR  THERMOSTATS  157 

with  double  walls  and  double  doors,  and  the  space  between  the  two 
walls  is  filled  with  water,  a  poor  conductor  of  heat,  which  tends  to 
keep  a  fairly  steady,  uniform  temperature  in  the  apparatus.  The 
source  of  heat  for  a  bacterial  incubator  is  now,  as  a  rule,  illuminating 
gas,  though  some  have  an  arrangement  which  will  permit  the  use  of 
a  coal-oil  lamp  in  places  where  gas  is  not  accessible.  The  most  modern 
incubators  use  electrical  appliances  as  a  source  of  heat. 

Thermoregulator. — In  order  to  keep  a  fairly  uniform  temperature 
in  a  bacterial  incubator  it  should  be  so  located  that  it  is  protected 
against  very  sudden  changes  of  temperature  and  must  be  provided 
with  a  thermoregulator.  The  latter  is  a  device  or  apparatus  which 
will  automatically  regulate  the  supply  of  heat.  Since  incubators 

FIG.  79 


Thermostat,  or  incubator. 

generally  receive  their  source  of  heaj;  from  illuminating  gas,  most 
thermoregulators  are  designed  to  control  the  gas  supply,  which  goes 
to  the  gas  flame  burning  underneath  the  thermostat.  The  temper- 
ature in  the  incubator  is  controlled  by  a  thermometer  projecting 
through  the  upper  double  wall  into  the  air  space  or  chamber  where 
the  cultures  are  kept.  The  flame  generally  used  under  a  thermostat 
is  a  so-called  micro-gas  lamp,  a  small  burner  which  furnishes  a  small, 
narrow  but  high  flame  protected  by  a  mica  cylinder.  Frequently  a 
Koch-Pfeil  safety  lamp  is  used.  This  is  so  constructed  that  it  will 
automatically  shut  off  the  gas  supply  if  the  flame  should  be  blown 
out  by  a  draft  of  air  or  for  some  other  reason.  However,  if  the  thermo- 
stat is  in  a  protected  place  this  danger  is  very  remote.  A  greater 
danger  comes  from  leaks  in  the  connecting  rubber  hose,  and  this  must, 


158 


PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 


therefore,  from  time  to  time,  be  inspected.  The  sense  of  smell  and  a 
lighted  candle  applied  carefully  along  the  tube  will  detect  any  leakage. 
The  thermoregulators  used  in  connection  with  thermostats  with 
gas  as  a  source  of  heat  are  generally  constructed  and  based  on  the 
following  principle  :  The  gas  is  led  into  the  upper  part  of  the  thermo- 
regulator  through  a  good  dense  hose,  connected  with  an  upper  tube 
of  the  instrument,  which  is  ground  into  the  lower  part.  The  vertical 
limb  of  the  T-shaped  upper  part  of  the  thermoregulator  is  drawn 


FIG.  80 


FIG. 


Incubator  thermometer. 


Thermoregulator. 


out  conically  and  has  an  open  tip  and  a  number  of  small  pinhole 
side  openings.  The  lower,  longer  tube  into  which  the  T-tube  fits  is 
filled  with  mercury  and  the  height  of  the  column  of  mercury  can  be 
regulated  by  a  screw  working  in  a  lateral  branch  of  the  lower  sealed 
glass  tube  or  mercury  receptacle.  If  the  column  of  mercury  closes 
the  lower  opening  of  the  T-tube  then  gas  can  escape  into  the  lower 
tube  only  through  the  pinhole  openings  of  the  T-tube.  The  lower 
glass  tube  or  receptacle  for  the  mercury  has,  well  above  the  level  of 
the  liquid,  a  lateral  tube  which  is  connected  with  the  micro-gas  lamp 
by  a  rubber  hose.  If  the  mercury  shuts  up  the  lower  end  of  the 
T-tube  very  little  gas  can  get  to  the  lamp,  and  it  burns  with  a  small 
flame.  If  the  mercury  recedes  from  the  lower  end  of  the  T-tube, 
gas  can  readily  entei  the  lower  glass  tube  and  the  gas  lamp  receives 
an  abundant  supply  and  burns  with  a  large  flame,  giving  much  heat. 
Starting  of  the  Incubator. — The  incubator  is  started  in  the  following 


manner : 


INCUBATORS  OR  THERMOSTATS  159 

1.  Immerse  the  thermoregulator  up  to  the  lower  lateral  branch, 
which  carries  the  regulating  screw  in  an  almost  completely  rilled 
flask.     With  the  thermoregulator  immerse  a  thermometer.     Heat  the 
water  in  the  flask  to  40°  C.,  over  a  small  flame  which  does  not  burn 
directly  under  either  the  thermometer  or  thermoregulator.      Now 
regulate  the  screw  on  the  lower  lateral  branch  so  that  the  mercury 
just  closes  the  lower  opening  of  the  T-tube. 

2.  Pour  water  heated   to   about   40°   C.   into   the   compartment 
between  the  double  walls  of  the  incubator.    This  is  done  through  an 
opening  on  top  of  the  incubator,  and  it  can  be  facilitated  by  using  a 
funnel.     When  the  compartment  is  completely  filled,  remove  the 
funnel  and  close  the  opening  with  a  cork  having  a  small  hole  in  the 
centre.    Provide  a  perforated  cork  or  rubber  stopper  for  the  thermo- 
regulator and  place  it  in  a  second  opening  near  the  margin  of  the 
incubator.     The  thermoregulator  now  dips  into  the  water  between 
the  walls  of  the  incubator. 

FIG.  82 


Koch's  safety  burner.  Micro-bunsen  burner. 

3.  Connect  the  T-tube  with  the  gas  pipe  and  the  median  lateral 
tube  with  the  micro-gas  lamp  by  a  rubber  hose.  Open  the  stopcock 
of  the  gas  pipe  and  light  the  micro-gas  lamp  under  the  incubator. 
The  latter  will  now  burn  with  a  small  flame,  because  the  thermo- 
regulator dips  into  water  about  40°  C.  and  the  mercury  shuts  up 
the  larger  opening  of  the  T-tube.  As  soon  as  the  water  in  the 
incubator  is  cooled  off,  the  mercury  recedes  and  the  lower  opening 
of  the  T-tube  being  now  free,  more  gas  goes  to  the  lamp.  If  the 
thermoregulator  has  been  set  to  40°  C.,  the  temperature  in  the 
chamber  of  the  incubator,  indicated  by  the  thermometer  which  pro- 
jects into  it,  is  generally  about  37°  C. 

Difficulties  in  Regulating  Incubators. — The  student  must  not  suppose 
that  it  is  a  very  easy  matter  to  regulate  a  thermostat  so  that  it  main- 
tains a  fairly  stationary  temperature.  Among  other  things  it  is 
necessary  to  regulate  the  stopcock  of  the  gas  pipe  so  that  the  gas 
pressure  in  the  thermoregulator  is  not  too  high.  This  pressure  in 
most  places,  as  well  known,  varies  very  much,  and  where  this  is 


160 


PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 


the  case  it  is  practically  impossible  to  keep  an  incubator  stationary. 
When  a  stationary  temperature  is  of  the  utmost  importance,  as,  for 
instance,  in  the  preparation  of  attenuated  cultures  to  be  used  as 
vaccines  (anthrax  vaccine),  the  gas  cannot  be  used  as  it  comes  from 
the  pipe,  but  must  first  be  led  into  a  gas-pressure  regulator  and  from 
there  to  the  thermoregulatoi .  Electric  thermoregulators  for  use 
in  connection  with  gas  lamps  have  also  been  constructed.  The  author 
has  not  yet  had  much  experience  with  the  electrical  thermostat,  where 
electricity  furnishes  the  source  of  heat,  and  he  does  not  know  whether 
these  are  more  reliable  than  the  incubators  heated  by  gas.  Incubators 
are,  of  course,  not  all  regulated  to  a  temperature  of  37°  C.;  they  are 
frequently  set  for  22°  C.,  and  for  special  purposes  at  other  temper- 
atures. 


FIG.  84 


FIG.  85 


Reichel  bacteriologic  filter:  a,  receptacle; 
b,  porcelain  filter;  c,  arm  for  vacuum  pump; 
d,  outlet. 


Pasteur  culture  filter. 


Bacteria  Filters. — In  order  to  separate  soluble  toxins  from  the  bac- 
teria which  have  produced  them  in  fluid  culture  media,  it  is  necessary 
to  filter  the  latter.  Even  the  best  and  densest  filter  papers  would 
not  do  for  this  purpose,  since  bacteria  can  pass  through  the  pores. 
Pasteur,  Chamberland,  Berkefeld,  and  others  have  devised  filter 
masses  which  are  so  dense  that  bacteria  cannot  pass  through  them. 
Kaolin,  clay,  and  other  similar  substances,  moulded  into  the  form 
of  bougies,  cylinders,  or  flasks,  are  the  masses  used.  They  are  not 
glazed,  of  course,  because  this  would  make  them  impervious  to  a 
watery  fluid.  Such  filters  vary  in  the  size  of  their  pores,  and  those 
having  the  smallest  will  not  permit  even  the  most  minute  micro- 
organisms to  pass.  The  Pasteur-Chamberland  filter  generally  has 
smaller  pores  than  the  Berkefeld  filter.  Whenever  a  fluid  medium 
in  which  bacteria  have  been  grown  is  filtered  for  the  purpose  of  obtain- 


BACTERIA  FILTERS 


161 


ing  a  bacteria-free  toxic  liquid  or  with  some  other  object  in  view  the 
filtrate  must  always  be  tested  to  find  out  whether  any  bacteria  are 
present.  This  is  done  by  inoculating  from  the  filtrate  solid  culture 


FIG.  86 


Apparatus  for  the  rapid  filtration  of  toxins,  etc.:  a,  filter  flask;  6,  Woulfe  bottle  to  guard 
against  regurgitation  of  water  from  the  pump;  c,  reservoir  for  the  filtrate;  d,  water  vacuum  pump. 
(McFarland.) 


FIG.  87 


FIG.  88 


Chamberland  filter. 


11 


Brass  suction  pump:  a,  connection  to  faucet;  d,  con- 
nection for  rubber  tube  from  filtering  apparatus; 
c,  valve  to  prevent  regurgitation  of  water  to  filter. 


162    PURE  CULTURES  FROM  PATHOLOGIC  MATERIAL 

media,  and  by  placing  part  of  it  in  the  incubator  to  see  whether  it 
will  become  cloudy  and  whether  anything  will  develop  in  it. 

Such  filtrates  are  commonly  said  to  be  germ-free  or  sterile.  The 
former  term  is  correct  provided  it  refers  to  visible  germs  of  known 
type  only.  The  term  sterile,  however,  is  objectionable,  because  it 
is  known  today  that  there  are  ultramicroscopic  invisible  filterable 
organisms  which  are  evidently  able  to  multiply  and  cause  diseases, 
such  as  hog  cholera,  rinderpest,  pleuropneumonia  of  cattle,  etc. 
Since  at  present  it  is  not  known  whether  bacteria-free  filtrates  may  not 
contain  other  ultramicroscopic  live  substances  which  so  far  cannot  be 
demonstrated  and  recognized,  the  term  sterile  should  not  be  used  in 
connection  with  filtrates. 

The  fluids  which  are  to  pass  through  Pasteur  or  other  filters  must 
be  subjected  to  pressure  because  gravity  alone  will  not  force  them 
through  rapidly  enough.  Therefore,  such  bacteria  filters  are  generally 
connected  with  a  suction  pump  screwed  to  a  faucet,  or  they  are 
attached  to  a  vacuum  apparatus  which  exhausts  the  air  by  a  steam 
or  gas  engine  or  electrical  device.  In  any  case  a  partial  vacuum 
is  formed  in  the  vessel  which  is  to  receive  the  filtrate,  and  the  external 
air  pressure  acting  upon  the  fluid  to  be  filtered,  presses  it  through 
the  pores  of  the  filtering  device.  It  is,  of  course,  understood  that  all 
parts  of  a  filtering  apparatus  must  be  connected  with  each  other  in  an 
absolutely  air-tight  manner;  otherwise,  filtration  is  not  perfect  and  the 
filtrate  may  become  contaminated  with  bacteria  from  aspirated  air. 


QUESTIONS. 

1.  Describe  the  method  of  obtaining  a  pure  culture  of  a  bacterium  suspected 
of  being  the  cause  of  an  abscess  which  has  not  yet  broken  through  or  ulcerated. 

2.  Describe  the  same  process  in  the  case  of  an  ulcerated  abscess. 

3.  What  is  meant  by  pouring  plates?    What  has  superseded  the  original  Koch 
method  of  pouring  plates  ? 

4.  Describe  a  Petri  dish.    How  are  these  prepared  for  use? 

5.  What  is  meant  by  naming  the  upper  end  of  a  culture  tube?    When  prac- 
tised? 

6.  Describe  methods  of  preparing  culture  tubes  in  order  to  pour  plates  from 
their  contents. 

7.  Describe  procedure  of  inoculating  a  set  of  three  tubes  from  which  plates 
are  to  be  poured. 

8.  How  are  the  Petri  dishes  treated  after  the  liquefied  agar  medium  has  been 
poured  into  them? 

9.  What  is  the  object  of  pouring  plates  (Petri  dishes)? 

10.  What  is  the  difference  in  development  of  colonies  in  Petri  dish  No.  1, 
No.  2,  and  No.  3,  respectively? 

11.  How  can  it  be  ascertained  when  several  different  types  of  colonies  are 
present;  which  is  the  causative  pathogenic  and  which  are  the  accidental  contam- 
inating microorganisms? 

12.  How  are  young  small  colonies  found  in  a  Petri  dish? 

13.  What  kind  of  contamination  is  frequently  found  in  Petri  dishes? 

14.  If  the  environment  makes  the  use  of  Petri  dishes  impossible,  how  can  a 
pure  culture  be  obtained  ? 

15.  What  is  Esmarch's  method  of  obtaining  pure  cultures? 

16.  What  is  an  impression  or  "Klatsch"  preparation? 


QUESTIONS  163 

17.  Describe  method  of  obtaining  a  pure  culture  from  bacteria  present  in  the 
circulating  blood  of  an  animal. 

18.  How  is  a  microscopic  preparation  made  from  fluid  culture  media  when 
only  a  few  microorganisms  are  thought  to  be  present? 

19.  Describe  a  bacteriologic  postmortem  examination  with  the  preparation  of 
cultures  from  the  heart's  blood  and  from  various  internal  organs. 

20.  What  is  meant  by  a  subculture  ?    What  other  term  for  subculture  is  in 
common  use? 

21.  Why  is  it  necessary  to  make  subcultures  frequently? 

22.  What  is  meant  by  the  first,  the  third,  the  twentieth  generation  of  a  patho- 
genic bacterium  ? 

23.  How  should  a  culture  be  labeled?    How  a  microscopic  stained  preparation? 

24.  What  is  a  streak  culture  ?    What  is  a  stab  culture  ? 

25.  How  is  a  "shake"  culture  prepared  and  for  what  purpose? 

26.  How  would  you  prepare  a  pure  culture  from  a  discharge  containing  glanders 
or  tubercle  bacilli?    Give  reasons  for  the  procedure  adopted. 

27.  What  is  an  anaerobic  culture? 

28.  What  are  the  principles  of  the  methods  used  in  preparing  anaerobic  cultures  ? 

29.  What  was  Koch's  first  method  to  exclude  atmospheric  air  from  a  plate 
culture  ? 

30.  How  is  the  stick  culture  method  practised  for  the  preparation  of  anaerobic 
cultures  ? 

31.  What  is  the  simplest  method  of  raising  anaerobic  bacteria  in  fluid  culture 
media  ? 

32.  Describe  the  method  of  using  a  Kipp  gas  generator  for  the  development  of 
hydrogen  gas. 

33.  What  precautions  are  necessary  in  mixing  water  and  sulphuric  acid  and 
why? 

34.  What  is  a  Novy  jar.    Describe  its  use  in  connection  with  a  Kipp  apparatus. 

35.  What  chemical  means  are  used  for  removing  the  oxygen  from  the  atmos- 
pheric air  in  a  closed  vessel? 

36.  How  are  these  chemical  means  made  use  of  for  a  single  culture  tube  and 
how  for  a  number  of  them  and  for  Petri  dishes? 

37.  How  is  an  anaerobic  culture  prepared  in  a  hen's  egg? 

38.  Describe  Wright's  method  of  preparing  anaerobic  cultures  in  completely 
filled  glass  tubes. 

39.  Describe  Wright's  modification  of  the  Buchner  method  of  raising  anaerobic 
cultures  in  tubes. 

40.  What  other  names  are  in  use  for  a  bacterial  brood  oven? 

41.  What  is  the  construction  of  such  apparatus? 

42.  What  is  a  micro-gas  lamp? 

43.  What  is  a  thermoregulator?    Describe  the  glass  mercury  thermoregulator. 

44.  How  is  it  adjusted  for  use  with  an  incubator  kept  at  37°  C? 

45.  Describe  the  method  to  start  an  incubator  to  be  kept  at  37°  C. 

46.  How  are  the  incubator  gas  hose  and  glass  tubes  inspected  for  leaks  ? 

47.  Under  what  circumstances  is  it  particularly  important  to  keep  the  thermo- 
stat always  at  a  uniform  temperature? 

48.  Describe  method  used  to  obtain  germ-free  filtrates  of  bacterial  cultures. 


CHAPTEK    XIII. 

IDENTIFICATION  OF  BACTERIA— CULTURAL  CHARACTERISTICS- 
ANIMAL  EXPERIMENTS. 

IN  practical  bacteriological  work  it  is  not  merely  sufficient  to 
obtain  a  bacterium  in  pure  culture  from  a  pathologic  product  or 
other  source,  but  the  organism  must  also  be  fully  identified.  This 
may  be  a  very  easy  matter,  requiring  perhaps  only  a  simple  animal 
experiment  or  a  simple  serum  test.  On  the  other  hand  it  may  require 
the  study  of  the  pure  culture  on  a  variety  of  media,  and  under  varying 
conditions,  with  careful  observation  of  the  naked-eye  appearances 
of  the  growth,  tests  for  metabolic  products  and  microscopic  exam- 
ination in  the  hanging  drop  and  the  examination  of  specimens  stained 
by  different  methods.  Certain  cultural  peculiarities,  like  the  lique- 
factioh  of  gelatin  and  the  production  of  acids  and  gases,  have 
already  been  considered.  It  is  also  necessary  to  note  the  effect  of 
certain  growing  bacteria  on  the  fluid  culture  media.  For  example, 
the  media  may  appear  clear,  with  the  formation  of  a  sediment  or 
cloudy,  either  slight  or  heavy.  It  should  also  be  observed,  when  a 
sediment  is  present,  whether  it  is  granular  or  not,  whether  a  pellicle 
is  formed,  and  whether,  in  the  latter  case,  streaks  from  it  reach  to 
the  bottom  of  the  tube  or  flask;  also,  whether  and  when  the  pellicle 
sinks  to  the  bottom.  The  precipitation  of  the  casein  in  milk  and  the 
tendency  of  certain  growths  on  a  solid  medium  to  adhere  to  the 
platinum  loop  when  introduced  must  also  be  noticed.  Any  and  all  of 
these  features  may  be  quite  characteristic  of  certain  bacteria,  and  may 
give  considerable  aid  in  identification.  The  colonies  on  plates  and 
slants  present  certain  characteristics  which  must  be  observed  in 
reflected  and  transmitted  light.  Their  size,  color,  dryness  or  moisture 
pigments  and  early  and  late  tendency  to  confluence  are  often  important 
characteristics.  A  number  of  descriptive  terms  applied  to  colonies 
and  bacterial  growth  as  a  whole  in  or  on  various  culture  media  must 
now  be  explained. 


CULTURAL  CHARACTERISTICS. 

Stab  Cultures. — Stab  cultures  in  gelatin  which  do  not  liquefy  the 
medium,  and  which  show  a  uniform  growth  along  the  stab,  without 
any  special  characters,  are  known  as  filiform  growths.  The  growth 
is  called  nodose  when  it  is  composed  of  closely  aggregated  colonies, 


STAB  CULTURES 


165 


and  beaded  when  the  loosely  placed  colonies  can  be  distinguished 
individually.  When  the  colonies  are  arranged  in  such  a  manner 
that  there  is  some  fancied  resemblance  to  papillary  excrescences  they 
are  known  as  papillate.  An  echinate  growth  indicates  one  beset  with 
sharp  extensions  which  radiate  from  the  centre  into  the  culture  medium. 
A  villous  growth  shows  some  resemblance  in  its  arrangement  to  the 
villi  of  the  intestines.  Arborescent  means  branched  like  a  tree,  and 
plumose  denotes  a  delicate  feathery  growth. 

When  liquefaction  occurs  in  the  gelatin  the  liquefied  zone  is  likely 
to  show  very  definite  arrangements  and  shapes,  to  which  the  following 
descriptive  terms  are  applied: 

FIG.  89 


Showing  characters  of  gelatin  stab  cultures:  A,  characters  of  surface  elevation:  1,  flat; 
2,  raised;  3,  convex;  4,  pulvinate;  5|  capitate;  6,  umbilicate;  7,  umbonate.  B,  characters  of 
growth  in  depth:  1,  filiform;  2,  beaded;  3,  tuberculate-ecinulate;  4,  arborescent;  5,  villous. 
(From  Chester.) 

Crateriform,  a  flat  excavation  like  a  saucer  or  crater. 

Tubular,  cylindrical,  saccate,  elongated  areas  of  liquefaction. 

Infundibular  or  conical,  more  or  less  funnel-shaped  areas  of  lique- 
faction. 

Fusiform  or  spindle-shaped,  those  which  have  the  greatest  diameter 
in  the  middle  and  taper  both  upward  and  downward. 

In  stratiform  liquefaction  the  whole  mass  at  the  upper  end  of  the 
gelatin  tube  becomes  fluid  and  the  process  progresses  downward, 
involving  deeper  and  deeper  strata  of  the  culture  medium. 

The  liquefied  gelatin  may  be  comparatively  clear,  with  a  sediment 


166 


IDENTIFICATION  OF  BACTERIA 


at  the  deepest  portion,  it  may  contain  flocculi,  it  may  be  uniformly 
cloudy  and  turbid,  and  it  may  finally  be  covered  by  a  pellicle. 

Shake  Cultures. — In  gelatin  shake  cultures,  gas  bubbles,  liquefaction, 
or  an  abundant  growth  toward  the  surface  are  distinguished.  The 
latter  indicates  an  aerobic  development,  while  an  abundant  growth 
in  the  lowest  strata  points  to  an  anaerobic  bacterium. 


FIG.  90 


Photographs  of  a  large  number  of  colonies  developing  in  a  layer  of  gelatin  contained  in  a 
Petri  dish.  Some  colonies  are  only  pinpoint  in  size;  some  are  as  large  as  a  pencil.  The  colonies 
here  appear  in  their  actual  size.  (Park.) 


Fio.  91 


FIG.  92 


Well-distributed  colonies  on  agar  in  Petri 
dish.     (Park.) 


Irregular  fringed  colony  (B.  malignant  edema). 
(From  Kolle  and  Wassermann.) 


Streak  Cultures. — In  streak  cultures  on  slanting  agar  or  blood-serum 
tubes,  filiform,  echinate,  beaded,  effuse,  and  arborescent  growths  are 


PLATES  167 

distinguished.  The  condition  of  the  condensed  water  in  the  tubes  is 
also  noted,  and  whether  it  is  clear  or  cloudy,  or  whether  there  is  a 
precipitate,  and  whether  the  latter  is  granular,  flocculent,  or  slimy,  or 
shows  any  other  particular  quality. 

Plates. — On  plates  are  studied  the  individual  colonies  in  particular, 
their  size  is  noted,  the  following  features  are  distinguished,  and  the 
following  descriptive  terms  are  used: 

Punctiform  denotes  the  dimensions  so  small  that  they  cannot  be 
well  measured  by  the  naked  eye. 

Round,  elliptical,  fusiform,  irregular  are  self-explanatory  terms; 
cochleate  is  a  colony  which  is  twisted  somewhat  like  the  shell  of  a  snail. 
.  Ameboid  is  a  colony  irregular  in  outlines  and  with  processes 
looking  somewhat  like  the  pseudopodia  of  an  ameba. 

Myceloid  is  a  colony  of  bacteria  with  radiating  slender  masses 
looking  more  or  less  like  a  mould  mycelium. 

FIG.  93  FIG.  94  FIG.  95 


I 


Round  surface  colony,  colon       Colony  of  typhoid  in  rather  Colonies    of    typhoid     and 

bacilli  grown  in    stiff   gelatin.  stiff  gelatin.     (Park.)  colon    bacilli    in    rather   soft 

(Park.)  gelatin.     (Park.) 

Filamentous  colonies  are  composed  of  an  irregular  mass  of  loosely 
woven  filaments. 

Floccose  indicates  a  dense  woolly  structure. 

Rhizoid  denotes  an  irregularly  branched,  root-like  character. 

Conglomerate  is  an  aggregation  of  small  colonies  more  or  less  equal 
in  size  and  character  which  present  one  compound  larger  colony. 

Toruloid  are  colonies  composed  of  several  small  round  or  oval 
colonies,  arranged  like  a  group  of  budding  torula  or  yeast  cells. 

Rosulate  means  shaped  like  a  rosette. 

Sometimes  terms  indicating  a  fancied  resemblance  to  certain  pic- 
torial objects  are  used;  anthrax  colonies,  for  example,  are  sometimes 
spoken  of  as  being  like  the  head  of  Medusa,  because  under  low  power 
they  show  tortuous  twisted  strings  and  masses  of  bacilli  at  the  margin 
which  somewhat  resembles  the  mythologic  serpent-surrounded  head 
of  a  Medusa.  With  reference  to  their  elevation  over  the  surface, 
colonies  are  described  as  flat,  raised,  convex,  pulvinate,  hemispheric, 
or  capitate;  also  umbilicated,  having  an  umbilicus-like  impression, 
and  umbonate,  having  a  central  nipple-like  elevation  or  knob. 


168 


IDENTIFICATION  OF  BACTERIA 
FIG.  96  FIG.  97 


Moist  raised  colonies  with  no  visible  structure, 
looking  like  a  drop  of  water. 


Deep  colonies,  usually  either  light 
brown,  gray,  or  yellow  in  coloi, 
opaque,  with  little  marking. 


FIG.  98 


FIG.  99 


FIG.   100 


a 


The  colonies  very  finely 
granular,  with  or  without 
twisted  threads  at  borders. 


Colony  in  gelatin.  The 
centre  is  coarsely  granular 
in  partly  fluid  gelatin. 
The  borders  are  formed  of 
wavy  bands  of  threads. 


Colonies  opaque  in  centre, 
with  lighter  borders.  The 
margin  is  coarsely  granular. 


FIG.  101 


FIG.   102 


FIG.   103 


Colonies  circular  in  form, 
composed  of  radiating 
threads. 


Colonies  with  opaque 
centres,  with  a  thin 
border  fringe. 


Colony  showing  a  network 
of  threads  which  is  thicker 
in  centre. 


Figs.  96  to  103  from  Lehman  and  Neumann. 


ANIMAL  EXPERIMENTS  169 

Surface  Details  and  Peripheral  Outlines. — In  addition  to  the  term 
smooth,  the  following  surface  details  and  peripheral  outlines  are  dis- 
tinguished. 

Alveolate,  marked  by  depressions  so  that  a  somewhat  honeycombed 
appearance  results. 

Punctate,  dotted  with  punctures  like  small  pinholes. 

Bullate,  irregular  elevations  somewhat  resembling  a  blistered  sur- 
face. 

Vesicular,  looking  like  small  vesicles,  and  due  to  gas  formation. 

Verrucose,  wart-like,  with  papillary  prominences. 

Squamous  or  scaly,  covered  with  scales. 

Echinate,  beset  with  pointed  prominences. 

Papillate,  beset  with  nipple-like  prominences. 

Rugose,  presenting  short,  irregular  folds,  in  consequence  of  the 
shrinkage  of  the  surface  growth. 

Corrugated,  arranged  in  long  folds. 

Edges. — The  edges  of  the  colonies  are  described  as  entire  when 
there  are  no  divisions  and  no  serrations;  as  undulate  when  the  outlines 
are  wavy;  as  repand  when  they  are  like  the  border  of  an  open  umbrella; 
as  ciliate  when  they  show  hair-like  extensions. 

Details. — The  finer  internal  details  of  the  colonies  are  studied  on 
microscopic  impression  preparations,  and  the  following  descriptive 
terms  are  used: 

A  reticulate  structure  shows  the  form  of  a  network  like  the  veins  of 
a  leaf. 

Areolate,  divided  into  rather  irregular  or  angular  spaces  by  more  or 
less  definite  boundaries. 

Gyrose,  marked  by  wavy  irregular  lines  like  the  convolutions  of 
the  brain. 

Marmorated,  marked  like  marble. 


ANIMAL  EXPERIMENTS. 

Animal  experiments  are  frequently  made  in  order  to  obtain  bac- 
teria in  pure  culture,  to  identify  pathogenic  bacteria  beyond  doubt, 
to  test  methods  of  disinfection  or  sterilization,  to  control  the  atten- 
uation of  vaccines,  to  obtain  immune  sera,  etc.  To  obtain  a  pure 
culture  of  glanders  from  an  open  lesion  due  to  the  Bacillus  mallei, 
it  is  generally  necessary  to  inoculate  a  male  guinea-pig  in  the 
particular  manner  described  in  the  chapter  on  glanders.  The  inocu- 
lation of  a  guinea-pig  or  a  mouse  makes  the  rapid  diagnosis  of  a 
doubtful  case  of  anthrax  possible;  similarly,  guinea-pigs  must  be 
inoculated  in  order  to  ascertain  whether  pasteurization  has  killed 
tubercle  bacilli  in  milk.  After  the  preparation  of  anthrax  vaccines  it 
is  necessary  to  estimate  accurately  the  attenuation  of  the  bacilli  by 
injecting  them  into  mice,  guinea-pigs,  and  rabbits.  When  testing 


170  IDENTIFICATION  OF  BACTERIA 

tetanus  and  diphtheria  antitoxins,  varying  proportions  of  antitoxin- 
toxin  mixtures  must  be  injected  into  guinea-pigs.  It  will  thus  be 
seen  that  numerous  circumstances  arise  in  practical  bacteriology 
when  the  animal  experiment  is  absolutely  necessary  and  unavoidable. 
Animal  inoculations  are  also  necessary  in  the  preparation  of  anti- 
toxins, of  antirabic  virus,  in  the  immunization  against  Texas  fever, 
rinderpest,  etc. 

In  conducting  experiments  on  animals  it  is  generally  necessary  first 
to  restrain  them.  Numerous  operating  tables,  both  large  and  small, 
with  restraining  devices  and  holders  for  mice  and  guinea-pigs  and 
larger  animals,  have  been  constructed.  Generally  elaborate  devices 
are  unnecessary,  and  a  few  boards  of  varying  sizes  with  four  nails 
or  blocks  driven  into  the  corners  will  suffice.  To  the  latter  the 
outstretched  legs  of  the  animal  are  fastened  with  twine.  The  prin- 
cipal instruments  used  in  inoculation  experiments  are  hypodermic 
syringes;  however,  operating  knives,  scissors,  forceps,  needles,  suturing 
material,  and  even  trephines  may  be  needed  in  certain  procedures. 
The  most  important  factor  in  all  animal  experiments  is  the  necessity 
of  strictest  asepsis,  in  order  that  accidental  and  misleading  results  may 
not  be  obtained.  Injection  of  a  bacterial  culture,  pathogenic  excretion, 
blood,  milk,  etc.,  into  an  animal  requires  shaving  and  thorough  anti- 
septic cleansing  of  the  part  to  be  inoculated.  The  inoculation  is 
made  with  a  sterile  syringe,  and  the  operator  must  work  with  clean, 
antiseptically  treated  hands. 

Subcutaneous  Inoculation. — Subcutaneous  inoculation  is  one  of  the 
most  common  methods  practised.  In  its  simplest  form  it  consists  of 
a  slight  incision  in  the  skin  without  any  aseptic  precautions,  followed 
by  rubbing  a  little  of  the  suspected  material  into  the  wound.  This 
method  is  sufficient  for  the  experimental  identification  of  anthrax  or 
bubonic  plague  bacilli.  In  other  instances  a  deep  pocket  must  be  made 
by  pushing  a  probe  under  the  skin  through  a  small  incision  and 
lifting  up  the  skin,  forming  thus  a  deep  protected  pocket.  This 
method  is  used  for  inoculating  material  suspected  of  containing 
tetanus  bacilli  or  spores. 

When  the  subcutaneous  method  is  used  with  asepsis  the  skin  must 
be  shaved  and  cleansed  and  a  sterile  hypodermic  needle  employed. 
In  using  hypodermic  syringes  the  operator  must  be  careful  not  to 
contaminate  himself  with  the  dangerous  bacteria  which  he  may  be 
handling.  A  good  syringe  with  a  tightly  closing  piston  should  be 
used.  Immediately  after  the  injection  the  entire  instrument,  including 
the  needle,  should  be  placed  in  a  vessel  with  water  that  is  slightly 
alkaline  and  boiled.  The  operator  must  then  thoroughly  cleanse  his 
hands  in  a  strong  bichloride  solution. 

Intravenous  Inoculation. — Another  method  of  inoculation  frequently 
used  is  the  intravenous  injection  in  which  the  material  is  injected 
directly  into  a  vein.  In  most  animals  the  jugular  can  generally  be 
used  for  this  purpose.  The  skin  over  it  is  shaved  and  cleansed  anti- 


QUANTITY  OF  CULTURE  INOCULATED  171 

septically  and  compression  is  made  central  to  the  point  of  injection. 
In  large  animals  it  may  be  advantageous,  first,  to  expose  the  vein  by 
an  incision  in  the  skin  and  fascia.  If  this  is  necessary,  it  must,  of 
course,  be  done  with  sterile  instruments.  Rabbits  are  generally 
inoculated  intravenously  through  the  large  veins  of  the  ear.  The 
posterior  vein  is  better  adapted  for  injections  than  the  larger  anterior. 
The  injection  is  made  from  the  external  surface  of  the  ear,  where  the 
hair  should  be  clipped  in  order  to  facilitate  sterilization  and  also  the 
finding  and  proper  compression  of  the  vein.  It  is  best  to  use  a  small, 
short  injection  needle.  Blood  may  also  be  withdrawn  by  this  same 
method.  This  is  necessary  in  cases  where  the  immunizing  serum  of 
a  treated  rabbit  is  tested  before  finally  killing  it  and  collecting  all  the 
serum. 

Intraperitoneal  Inoculation. — This  is  very  frequently  practised  in 
bacteriologic  tests.  In  addition  to  the  usual  asepsis,  it  is  important  to 
make  the  injection  in  such  a  manner  as  not  to  injure  the  intestines. 
This  may  be  accomplished  by  either  one  of  two  methods.  One  is  by 
the  use  of  dull  needles,  which  require  a  preliminary  incision  through 
the  skin  and  fascia  in  the  median  line  of  the  anterior  abdominal  wall. 
The  needle  is  then  pushed  right  in  the  median  line  through  the  peri- 
toneum and  the  overlying  tissues.  In  the  other  method  the  animal 
is  placed  hind  legs  upward,  a  position  in  which  the  intestines  fall 
toward  the  diaphragm.  If  the  needle  is  introduced  in  the  middle  line 
below  the  umbilicus,  and  held  very  obliquely  so  that  it  does  not  pene- 
trate very  far  into  the  abdominal  cavity,  the  danger  of  injuring  the 
intestines  or  any  of  the  abdominal  viscera  is  reduced  to  a  minimum. 

Other  Forms  of  Inoculation. — Inoculations  are  occasionally  made  in 
the  thoracic  cavity  through  an  intercostal  space,  but  more  frequently 
in  the  anterior  chamber  of  the  eye,  where  the  developing  lesions  can 
be  studied  directly  from  day  to  day.  Subdural  inoculations  are  made 
after  preliminary  incision  in  the  scalp  and  trephining  of  the  skull. 
Injections  into  the  spinal  canal  may  be  made  in  the  lumbar  region, 
by  inserting  a  long  needle  of  the  hypodermic  syringe  into  the  canal, 
between  two  vertebrae,  laterally  from  the  median  line. 

Infections  of  the  Intestinal  tract  are  made  by  feeding  animals  with 
the  infected  material.  If  it  is  desirable  to  introduce  such  material 
directly  into  the  intestines,  it  is  necessary  to  perform  a  laparotomy, 
exposing  the  duodenum,  into  which  the  injection  is  made  directly 
with  a  very  small  hypodermic  syringe. 

Animals  are  sometimes  infected  experimentally  through  the  res- 
piratory tract.  This  is  done  by  connecting  their  cages,  directly  or 
indirectly,  with  a  spraying  apparatus  which  disseminates  the  infected 
material. 

Quantity  of  Culture  Inoculated. — It  is  generally  desirable,  except  in 
purely  diagnostic  work,  to  use  a  definite  quantity  of  the  pure  culture 
for  inoculation.  This  may  be  accomplished  by  a  variety  of  methods: 
(1)  A  pure  culture  is  prepared  by  inoculating  10  c.c.  of  bouillon. 


172  IDENTIFICATION  OF  BACTERIA 

This  is  kept  in  the  incubator  for  twenty-four  hours,  then  well  shaken, 
and  a  definite  fraction  of  the  whole  amount  used  for  each  animal. 
(2)  An  agar  tube  with  a  slanting  surface  is  inoculated  by  rubbing  the 
material  thoroughly  over  the  entire  surface  writh  the  platinum  loop. 
After  incubating  for  twenty-four  hours  the  whole  growth  is  removed 
with  the  platinum  loop  and  intimately  and  uniformly  mixed  with  lO^.c. 
of  an  0.85  per  cent,  salt  solution,  a  fraction  of  which  is  finally  inocu- 
lated into  the  experimental  animals.  (3)  A  platinum  loop  of  definitely 
known  size  is  used  for  removing  a  portion  of  the  growth  from  an 
agar  slant.  One  holding  just  2  mg.  of  a  bacterial  growth  is  known 
as  a  "Normaloese,"  or  "Normal  loop."  To  make  these  loops  a  little 
apparatus  consisting  of  a  number  of  steel  rods  held  in  small  wooden 
blocks  has  been  constructed.  By  winding  the  free  end  of  the  platinum 
wire  around  the  smallest  steel  rod  a  loop  is  formed  which  will  just 
hold  2  mg. ;  the  other  steel  rods  form  loops  holding,  respectively,  2, 
5,  and  10  mg.  The  quantity  of  bacterial  growth  removed  from  an 
agar  slant  with  such  a  "Normaloese"  is  well  rubbed  up  with  2  c.c.  of 
physiologic  salt  solution  and  the  entire  mixture  is  injected.  An  animal 
so  treated  is  said  to  have  received  one,  two,  or  five,  as  the  case  may  be, 
"Normaloesen"  of  a  certain  bacterial  growth. 

Collodion  Sacs. — It  is  sometimes  desirable  to  implant  bacteria  into 
the  body  of  an  animal  in  such  a  manner  that  they  are  protected 
against  the  phagocytes  of  that  animal.  This  is  done  by  the  aid  of 
collodion  sacs  whose  walls  permit  osmotic  processes  to  continue  but 
prevent  the  emigration  of  bacteria  and  the  entrance  of  leukocytes  and 
other  cells.  The  simplest  method  for  preparing  them  is  as  follows: 
Clean  a  small  test-tube  and  dry  it  completely  by  washing  first  in 
absolute  alcohol  and  then  in  ether.  Pour  some  fairly  thick  collodion, 
or  celloidin  as  used  in  section  work,  into  the  dry  tube  and  move  it 
continually  in  such  a  manner  that  the  collodion  coats  its  entire  interior. 
When  the  collodion  becomes  very  thick  in  consequence  of  evaporation, 
let  the  last  few  drops  run  over  the  rim  at  the  mouth  to  the  outside  of 
the  tube.  The  tube  is  then  filled  with  water,  and  after  a  little  while 
the  outside  collodion  is  peeled  off  without  tearing  it  from  its  connection 
with  the  inside  collodion.  The  collodion  surrounding  the  inner  mouth 
of  the  tube  is  loosened,  and  by  allowing  water  from  the  faucet  to  flow 
between  it  and  the  test-tube  wall  it  gradually  separates.  A  slight 
pulling  on  the  collodion  is  often  necessary  completely  to  detach  it 
from  the  tube.  A  glass  tube  slightly  smaller  than  the  test-tube  is  now 
prepared,  and  near  one  end  a  narrow  constriction  is  blown  in  over  a 
flame.  About  1 J  inches  of  the  lower  end  of  the  collodion  sac  is  now  cut 
off  and  the  open  end  is  slipped  over  the  constricted  glass  tube  and 
fastened  to  it  with  a  piece  of  good  surgical  silk.  Finally,  fresh  thick 
collodion  is  painted  over  the  silk  and  the  upper  rim  of  the  sac  over 
the  glass  tube,  making  a  water-tight  connection  between  the  sac  and 
glass  tube.  Of  course,  each  sac  must  be  tested  before  it  is  slipped 
over  the  glass  tube.  A  number  of  them  should  be  prepared,  as  only 


QUESTIONS  173 

a  portion  will  be  of  such  quality  that  they  can  be  used.  The  collodion 
sacs  are  then  filled  with  nutrient  bouillon  and  placed  in  larger  test 
tubes  containing  the  same  bouillon.  Both  the  glass  tube  and  the 
test  tube  are  cotton  plugged,  and  then  sterilized  and  cooled.  Later 
the  collodion  sacs  can  be  inoculated  with  the  platinum  rod.  They 
must  then  be  taken  out  of  the  tubes  and  dried  externally  with  sterile 
cotton  and  the  constriction  heated  over  a  flame,  drawn  out  and 
securely  sealed.  They  are  now  ready  for  implantation  into  the  peri- 
toneal cavity  of  an  animal. 

Non-pathogenic  bacteria  in  collodion  sacs  implanted  in  the  peri- 
toneal cavity  of  an  animal  can  be  so  changed  that  they  acquire 
pathogenic  properties,  and  after  removal  from  the  animal  and  sub- 
sequent inoculation  in  another  animal  of  the  same  species  they  will 
produce  disease  because  they  are  able  to  multiply  and  resist  phago- 
cytosis. 

QUESTIONS. 

|1.  How  can  a  bacterium  obtained  in  pure  culture  from  some  pathologic 
product  be  fully  identified? 

2.  What  are  the  different  changes  in  appearances  produced  by  bacteria 
(a)  in  nutrient  bouillon  ?     (b)  in  milk  ? 

3.  Explain  the  meaning  of  the  following  terms  used  with  reference  to  a  non- 
liquefying  growth  of  a  bacterium  in  gelatin :    Filiform,  nodose,  beaded,  papillate, 
echinate,  villous,  arborescent,  plumose. 

4.  Explain  the  meaning  of  the  following  terms,  used  with  reference  to  the 
liquefying  growths  in  gelatin :     Crateriform,  tubular,  cylindrical,  saccate,  infun- 
dibular, conical,  fusiform,  stratiform  liquefaction. 

5.  What  features  should  be  noted  in  a  gelatin  shake  culture? 

6.  What  features  should  be  noted  in  the  condensed  water  of  an  agar  slant? 

7.  What  arrangement  offers  the  best  chances  to  study  the  characteristics  of 
individual  colonies? 

8.  Explain  the  following  terms  used  in  the  description  of  bacterial  colonies: 
Round,  elliptical,  fusiform,  irregular,  cochleate,  ameboid,  myceloid,  filamentous, 
floccose,  rhizoid,  conglomerate,  toruloid,  rosulate. 

9.  What  is  meant  by  a  colony  "resembling  the  head  of  a  Medusa?"     Name 
a  bacterium  forming  such  colonies. 

10.  What  terms  are  used  with  reference  to  the  different  types  of  elevations 
of  colonies  over  the  surface  of  the  culture  medium? 

11.  What   are   the   meanings  of   the  following   terms:     Alveolate,  punctate, 
bullate,  vesicular,  verrucose,  squamous,  echinate,  papillate,  rugose,  corrugate? 

12.  What  terms  are  used  in  the  description  of  the  margins  of  colonies? 

13.  Explain  the  following  terms:   Reticulate,  areolate,  gyrose,  marmorated. 

14.  For  what  purposes  are  animal  experiments  made  in  bacteriologic  studies. 

15.  Describe  the  subcutaneous  method  of  inoculation. 

16.  The  intraperitoneal  method. 

17.  The  intravenous  method. 

18.  The  subdural  method.    The  method  of  inoculating  into  the  spinal  canal. 

19.  How  is  a  small  animal  restrained  for  inoculation? 

20.  What  precautions  are  used  to  avoid  injuries  in  intraperitoneal  inoculation? 

21.  What  is  a  "Normaloese ?"    What  is  the  object  of  using  it? 

22.  What  other  methods  are  used  to  inoculate  a  definite  amount  of  a  bacterial 
growth  ? 

23.  Describe  the  method  of  preparing  collodion  sacs. 

24.  What  is  the  object  of  using  them? 


CHAPTEE    XIV. 

METHODS  OF  EXAMINING  AIR,  SOIL,  WATER  AND  OTHER 
FLUIDS  FOR  BACTERIA. 

THE  general  principle  underlying  the  examination  of  air,  soil, 
water,  and  other  fluids  is  that  of  mixing  a  definite  amount  of  these 
substances  with  a  suitable  culture  medium,  pouring  plates  or  Petri 
dishes,  and  studying  the  developing  colonies  of  bacteria,  yeast  cells, 
moulds,  etc.,  as  to  species  and  numbers  of  colonies  developed.  In 
the  case  of  substances  like  soil,  water,  milk,  and  other  fluids  a  small 
definite  amount  can  be  taken  directly  and  mixed  with  the  culture 
medium.  In  the  case  of  the  air,  a  known  volume  must  be  aspirated 
either  into  a  culture  medium  or  into  a  sterile  bland  substance  which 
is  subsequently  mixed  with  a  culture  medium. 

The  methods  of  examination,  however,  vary  a  good  deal  according 
to  the  particular  object  in  view.  If  a  soil  is  to  be  examined  for  the 
presence  of  tetanus,  anthrax  or  malignant  oedema  bacilli,  some  of 
the  material  is  inoculated  directly  into  susceptible  animals  (mice, 
guinea-pigs,  etc.)  without  first  resorting  to  cultural  methods.  Like- 
wise, in  the  examination  of  air  for  tubercle  bacilli  (first  extensively 
carried  on  by  Cornet)  the  dust  which  is  aspirated  with  the  air  or 
which  has  settled  spontaneously  from  it  must  be  secured  and  inocu- 
lated directly  into  guinea-pigs. 

FIG.  104 


Wolffhtigel's  apparatus  for  counting  colonies. 

Counting  the  Colonies. — In  the  examination  of  air,  soil,  water,  milk, 
etc.,  for  microorganisms  (bacteria,  yeast  cells,  moulds,  etc.)  it  is  often 
desirable  to  obtain  as  exact  a  numerical  estimate  as  possible.  This 
is  accomplished  after  proceeding  as  indicated  above  by  counting  the 
number  of  colonies  developed  in  the  Petri  dishes. 

Wolffhugel's  Counting  Apparatus. — Colonies  are  generally  counted 
with  the  aid  of  a  Wolffhugel  counting  apparatus,  which  consists  of  a 


BACTERIOLOGIC  EXAMINATION  OF  AIR  175 

black  glass  plate,  contained  in  a  wooden  frame.  The  Petri  dish  is 
placed  on  the  black  glass.  Above  it,  resting  on  four  blocks,  is  a 
transparent  glass  plate  which  has  been  ruled  with  a  diamond  into 
square  centimeters  and  fractions  of  a  square  centimeter.  The  colonies 
in  one  cubic  square,  both  on  the  surface  and  in  the  depth  of  the  medium 
are  counted  with  the  aid  of  a  hand  magnifying  glass  or  the  low-power 
lens  of  a  compound  microscope  with  a  large  stage.  This  is  done  for 
a  number  of  squares  and  the  average  number  of  colonies  per  square 
centimeter  is  then  calculated.  If  it  has  not  been  done  previously, 
the  exact  diameter  of  the  lower  part  of  the  Petri  dish  is  then  measured 
and  the  surface  of  the  culture  medium  contained  in  the  lower  dish  is 
calculated  according  to  the  formula  for  the  surface  of  a  circle,  which 
is  r2  7i.  For  example :  Counting  5  square  centimeters : 

Square  No.  1  .  |    ,,;   J   Q-  •  •• 16  colonies 

Square  No.  2  ...."..,.  , 23  colonies 

Square  No.  3  .      .      .      .      :.      ..'.'.      .      .      .       '.'""  . '  '  •.'      .  14  colonies 

Square  No.  4  .      .      J '•.''•';  '  .      .rvv1*. 18  colonies 

Square  No.  5  ..  ..„    ..*;,..,>.  .,.[>  .;,..,,  •  (<*•<-!{'    ,j[i  •,<<,•.''•       •  19  colonies 

Total '".'"'."".''.     90  colonies 

Average  per  square  cm.     .    t-.    t  ,/f  ^  j    ?>      .      .      .       .      -j '    •      \  '   •      18  colonies 

Diameter  of  lower  part  of  Petri  dish       .'.-.......      .        6  om. 

Hence  radius     .' '.''".    ''.''.  >;i!'». '".''   .'  ' />!V    ';'    I.1'"!.;'<  >C{     .        3cm. 
Hence  surface  9X3.14       »,;,-    ••>,].  ».-:«t!rt'     f  ,r?v    «v!«!      •      .;,[.      28.26  cm. 
This  figure  multiplied  by  18  equals 308.68  colonies 

This  means  that  the  Petri  dish  has  developed  309  colonies;  and  if  1  c.c.  of  water  was 
mixed  with  the  culture  medium,  the  result  shows  that  this  water  did  contain  309  live  bacteria 
per  cubic  centimeter. 

Esmarch's  Apparatus. — Sometimes,  in  the  examination  of  air, 
water,  etc.,  plates  cannot  be  poured.  In  such  cases  Esmarch  role- 
tubes  or  other  glass  tubes  coated  on  the  inside  with  the  culture  medium 
must  be  prepared  for  the  development  of  the  colonies.  With  tubes 
the  Wolffhiigel  counting  apparatus  is  replaced  by  a  special  magni- 
fying glass  with  tube  holder,  devised  by  Esmarch.  The  outside  of 
the  glass  tube  is  divided  into  a  number  of  equal  fields.  The  colonies 
in  a  number  of  fields  are  counted,  the  average  is  obtained  and  multi- 
plied by  the  total  number  of  fields.  In  other  words,  the  fields  are 
treated  the  same  as  the  squares  in  the  preceding  example. 

These  methods  furnish  an  approximate  result  only,  but  one  that  is 
accurate  enough  for  the  purpose. 

Bacteriologic  Examination  of  Air. — When  a  very  exact  quantitative 
bacteriologic  examination  of  air  is  unnecessary  the  method  practised 
by  Robert  Koch  will  be  found  both  simple  and  satisfactory.  Culture 
media  are  poured  into  Petri  dishes  and  allowed  to  solidify.  The  lid 
of  the  dish  is  then  removed  and  the  culture  medium  exposed  to  the 
air  for  a  definite  period  of  time  (for  instance,  ten  or  fifteen  minutes). 
The  lid  is  then  replaced  and  the  Petri  dish  is  kept  at  room  temperature 
or  incubated  in  the  usual  manner.  By  this  simple  method  compara- 


176     EXAMINING  AIR,  SOIL,  WATER,  AND  FLUIDS  FOR  BACTERIA 

live  studies  of  the  air  can  be  made  in  the  open,  in  a  room,  in  a  barn, 
in  a  basement,  etc.,  as  the  difference  in  the  number  of  colonies  devel- 
oped in  Petri  dishes  exposed  simultaneously,  or  approximately  so,  to 
air  under  different  conditions  for  the  same  period  of  time,  gives  a 
fairly  accurate  indication  of  the  variations  in  the  bacterial  contents 
of  the  air.  Data  as  to  the  relation  between  the  number  of  bacteria 
and  moulds  present  can  also  be  obtained  by  this  method. 

EXACT  METHOD. — For  the  exact  quantitative  estimation  of  bac- 
teria and  moulds  a  variety  of  methods  have  been  devised.  The 
simplest  of  these,  which  is,  however,  only  slightly  more  accurate  than 
the  one  first  described,  is  as  follows.  A  large  Erlenmeyer  flask,  prefer- 
ably one  holding  at  least  2  liters  (2000  c.c.),  is  filled  with  water,  cotton 
plugged,  and  thoroughly  sterilized  in  the  steam  sterilizer.  After 
being  removed  from  the  latter  the  water  is  cooled  and  the  flask  taken 
to  the  place  where  the  air  is  to  be  examined.  A  test  tube  containing 
25  to  30  c.c.  of  agar  or  gelatin  is  heated  in  a  water  bath  and  cooled 
down  to  near  the  point  of  solidification.  When  this  is  ready  the  cotton 
plug  is  removed  from  the  flask  and  the  sterile  water  poured  out, 
which  is  now,  of  course,  replaced  by  two  liters  of  air.  The  flask 
must  be  energetically  shaken  with  the  mouth  down  in  order  to  get 
it  as  dry  as  possible.  It  is  then  placed  on  a  level  surface.  The  melted 
culture  medium  poured  in,  the  cotton  plug  replaced,  and  the  medium 
allowed  to  solidify.  The  flask  must  be  kept  perfectly  quiet,  either 
at  room  or  incubator  temperature,  which  causes  the  microorganisms 
contained  in  the  air  to  fall  to  the  bottom  and  develop  into  colonies 
on  the  culture  medium.  Before  using  the  latter  for  this  purpose  it 
should  have  been  kept  in  the  incubator  for  several  days,  so  that  all 
of  the  water  of  condensation  has  evaporated  and  the  medium  is 
comparatively  dry.  Otherwise,  the  water  of  condensation  is  squeezed 
out  of  the  solidifying  medium  and  running  over  its  surface  will 
interfere  with  the  formation  of  the  proper  number  of  colonies.  After 
remaining  in  the  incubator  for  a  few  days  the  flask  is,  taken  out  and 
the  colonies  which  have  developed  in  the  medium  on  the  bottom  can 
be  counted  with  a  magnifying  glass.  More  exact  methods  for  counting 
the  number  of  bacteria  and  moulds  in  the  air  are  the  following: 

HESSE'S  METHOD. — The  apparatus  consists  of  a  glass  tube  70  cm. 
long  and  3.5  cm.  wide,  the  interior  of  which  is  coated  with  a  thin 
layer  of  gelatin.  One  end  of  the  tube  is  closed  by  a  solid  rubber 
stopper  or  cap,  the  other  by  a  perforated  rubber  cork  which  carries 
a  small  glass  tube.  This  latter  is  connected  by  a  rubber  hose  with  a 
suction  bottle  or  flask  which  is  in  turn  connected  with  a  second  flask 
of  the  same  type,  each  of  exactly  2  liters'  capacity.  After  the  long 
glass  tube  has  been  properly  sterilized  in  the  hot-air  sterilizer  and 
coated  with  gelatin  it  is  mounted  in  a  horizontal  position  and  one 
of  the  suction  flasks  is  connected  with  the  small  glass  tube  in  the 
perforated  rubber  stopper.  This  flask  is  filled  with  water  and  con- 
nected with  the  second  suction  flask,  which  is  empty,  in  the  manner 


BACTERIOLOGIC  EXAMINATION  OF  AIR  177 

shown  in  Fig.  105.  The  rubber  cap  is  then  removed  from  the  end  of 
the  horizontal  tube  and  enough  suction  by  mouth  made  at  the  exit 
tube  of  the  lower  and  empty  flask  to  start  the  water  running  from 
the  upper  flask.  The  water  then  siphons  out  of  the  higher  flask  into 
the  lower  one,  and  during  this  process  two  liters  of  air  are  aspirated 
through  the  gelatin  coated  tube.  The  outer  end  of  the  tube  is  then 
closed  with  the  rubber  cap  and  the  suction  flasks  reversed.  The  cap 
is  again  removed,  and  as  suction  is  made  on  the  empty  flask  the  water 
flows  as  before  from  the  upper  to  the  lower  flask  and  two  more  liters 
of  air  are  aspirated  through  the  gelatin-coated  tube.  The  operation 
is  repeated  a  number  of  times  until  about  20  liters  of  air  have  been 

FIG.  105 


Hesse's  apparatus  for  collecting  bacteria  from  the  air.     (McFarland.) 

aspirated  The  long  tube  is  then  disconnected,  closed  at  both  ends 
with  sterile  cotton  plugs,  and  kept  in  a  horizontal  position  for  several 
days  to  permit  the  colonies  to  develop.  These  are  then  counted. 
During  the  aspiration  the  water  should  flow  at  such  a  rate  that  1  liter 
passes  from  the  upper  to  the  lower  flask  in  one  to  two  minutes. 

EYRE'S  METHOD. — Eyre  recommends  the  following  apparatus  and 
method  of  quantitative  bacteriologic  examination  of  air: 

Apparatus. — 1.  Aspirator  bottle,  10  liters'  capacity,  fitted  with  a 
delivery  tube,  and  having  its  mouth  closed  with  a  perforated  rubber 
stopper,  through  which  a  short  length  of  glass  tubing  passes. 

2.  Erlenmeyer  flask,   250  c.c.   capacity  (having  a  wide  mouth, 
properly  plugged  with  cotton),  containing  50  c.c.  sterile  bouillon. 
12 


178-     EXAMINING  AIR,  SOIL,  WATER,  AND  FLUIDS  FOR  BACTERIA 

3.  Rubber  stopper  to  fit  the  mouth  of  the  flask,  perforated  with  two 
holes,  and  fitted  as  follows:  Take  a  piece  of  glass  tubing,  15  cm.  in 
length  and  bend  up  3  cm.  at  either  end  at  right  angles  to  the  main 
length  of  tubing.  Pass  one  of  the  bent  ends  through  one  of  the  per- 
forations in  the  stopper;  plug  the  opposite  end  with  cotton.  Take 
a  glass  funnel,  5  or  6  cm.  in  diameter,  with  a  stem  15  cm.  long,  and 
bend  the  stem  close  up  to  the  apex  of  the  funnel  in  a  gentle  curve 
through  a  quarter  of  a  circle.  Pass  the  long  stem  through  the  other 
perforation  of  the  rubber  stopper.  In  addition,  rubber  tubing,  screw 
clamps  and  spring  clips  for  tubing,  a  steam  sterilizer,  retort  stand  and 
clamps  are  required. 


FIG.   106 


Arrangement  of  apparatus  for  air  analysis.     (Eyre.) 

Method  of  Procedure. — 1.  Fill  the  aspirating  bottle  with  10  liters 
of  water  and  attach  a  piece  of  rubber  tubing  with  a  screw  clamp  to 
the  delivery  tube.  Regulate  the  screw  clamp  by  actual  experiment 
so  that  the  tube  delivers  1  c.c.  of  water  per  second.  At  this  rate  the 
aspirator  bottle  is  emptied  in  just  under  three  hours.  Close  the  rubber 
tube  below  the  clamp  by  means  of  a  spring  clip,  and  make  up  the 
contents  of  the  aspirator  bottle  to  10  liters. 

2.  Sterilize  the  fitted   rubber  cork  with  its  funnel  and  tubing  by 
boiling  in  the  steam  sterilizer  for  ten  minutes. 

3.  Remove  the  cotton  plug  from  the  250  c.c.  Erlenmeyer  flask 
containing  50  c.c.  sterile  bouillon  and  replace  it  by  the  rubber  stopper 
with  its  funnel  and  bent  tube.     Make  sure  that  the  end  of  the  stem 
of  the  funnel  is  immersed  in  the  bouillon. 


BACTERIOLOGIC  EXAMINATION  OF  AIR  179 

4.  Place  the  Erlenmeyer  flask  with  the  bouillon  in  a  glass,  metal, 
or  other  suitable  vessel,  and  pack  it  around  with  cracked  ice.    Place 
the  Erlenmeyer  flask  on  a  stand  or  box  so  that  the  bent  glass  tube 
in  the  perforated  stopper  can  be  conveniently  connected  by  a  rubber 
tube  with  the  aspirating  bottle. 

5.  Remove  the  spring  clip  from  the  rubber  tube   and  allow  the 
water  to  run  from  the  aspirating  bottle  at  the  rate  of  1  c.c.  a  second, 
as  previously  arranged. 

6.  From  time  to  time  replace  the  ice  in  order  to  keep  the  bouillon 
near  0°  C.  in  order  to  prevent  multiplication  of  the  bacteria  in  it. 

6.  When  the  10  liters  of  water  have  run  out  of   the   aspirating 
bottle,  a  corresponding  quantity  of  air  has  been  aspirated  through  the 
50  c.c.  of  bouillon  in  the  Erlenmeyer  flask.    The  bouillon  now  con- 
tains all  the  bacteria  that  were  originally  in  the  10  liters  of  aspirated 
air.    Then  disconnect  the  Erlenmeyer  flask  with  the  50  c.c.  of  bouillon 
and  shake  the  contents  well. 

7.  In  the  meantime  a  number  of  gelatin  or  agar  tubes  have  been 
liquefied  and  cooled  down  to  near  the  point  of  solidification  of  the 
media.    Then  with  sterile  graduated  pipette  add  some  of  the  bouillon 
to  the  fluid  culture  media  in  these  tubes.    To  the  first  add  0.5  c.c., 
to  the  next  0.3  c.c.,  and  to  a  third  0.2  c.c.    The  contents  of  the  tubes 
are  next  poured  into  two  sets  of  sterile  Petri  dishes,  one  of  which  is 
kept  at  room  and  the  other  at  incubator  temperature.    After  a  few 
days  the  colonies  are  counted  and  the  average  per  cubic  centimeter 
of  bouillon  is  calculated.    This  average  multiplied  by  fifty  indicates  the 
number  of  bacteria  contained  in  the  50  c.c.  of  bouillon  in  the  Erlen- 
meyer flask  and  represents  the  bacteria  present  in  the   10   liters  of 
air  which  have  been  aspirated  through  the  apparatus.    The  number 
of  bacteria  present  in  air  is  usually  stated  in  terms  of  cubic  meters, 
and  since  a  cubic  meter  is  equal  to  1000  liters,  the  last  figure  must 
be  multiplied  by  100  to  obtain  the  number  of  bacteria  in  one  cubic 
meter. 

METHODS  OF  FRANKLAND  AND  PETRI. — Frankland  and  Petri  have, 
independent  of  each  other,  devised  a  method  in  which  the  air  is  first 
aspirated  into  a  filter  of  sterile  quartz  sand.  The  grains  of  sand  have 
an  average  diameter  of  ^  to  -J  mm.,  and  are  contained  in  a  piece  of 
glass  tubing  6  to  10  cm.  long  and  about  2  cm.  wide.  The  sand  is 
held  in  the  centre  of  the  tube,  and  divided  into  two  equal  portions  by 
fine  wire  gauze.  One  end  of  the  tube  is  closed  by  a  cotton  plug  and 
the  other,  after  thorough  dry  sterilization,  by  a  perforated  rubber 
stopper  containing  a  small  glass  tube.  When  the  device  is  to  be 
used  the  latter  is  connected  with  an  air  pump.  The  tube  containing 
the  sand  filter  is  held  upright  during  the  process  of  aspiration  which 
is  continued  at  the  rate  of  10  liters  per  minute  for  from  ten  to  twenty 
minutes.  The  upper  and  lower  portions  of  sand  are  then  separately 
mixed  with  liquefied  gelatin  or  agar,  well  shaken,  and  poured  into 
Petri  dishes.  The  lower  portion  of  sand  should  be  found  sterile,  the 


180     EXAMINING  AIR,  SOIL,  WATER,  AND  FLUIDS  FOR  BACTERIA 

upper  portion  alone  developing  colonies.  These  are  counted  in  the 
usual  manner. 

Modifications  of  the  above  method  have  been  introduced  by  Ficker, 
who  uses  glass  powder  as  the  filter  mass,  and  by  Frankland,  who 
uses  sterile  powdered  sugar. 

Sedgwick  and  Tucker  have  modified  the  filtering  device  by  pro- 
viding at  the  upper  end  an  expanded  portion  ruled  outside  in  equal 
squares.  After  aspiration  of  the  air,  gelatin  is  poured  directly  into  the 
tube.  As  soon  as  the  sugar  powder  loaded  with  the  air  bacteria  is 
dissolved  the  tube  is  rolled  on  ice  and  becomes  an  Esmarch  roll-tube. 
This  method  avoids  the  transfer  of  the  filter  material  into  a  separate 
plate  or  tube. 

Bacteriologic  Examination  of  Water. — Collection  of  Water. — The 
collection  of  water  differs  according  to  the  source  from  which  it  is 
obtained.  Two  factors  must  be  particularly  considered:  (1)  Care 
must  be  exercised  that  the  water  running  into  a  sterile  flask  does 
not  wash  into  it  bacteria  which  may  have  been  on  the  outside  of 
the  mouth  of  the  vessel.  The  risk  can  easily  be  avoided  by  sterilizing 
the  flasks  in  the  dry  air  sterilizer  and  wrapping  them  in  paper  which 
is  not  removed  until  the  actual  moment  of  use.  (2)  Care  must  be 
taken  that  water  coming  from  a  faucet  does  not  collect  from  the 
mouth  of  the  latter  bacteria  which  may  have  been  deposited  here  by 
some  means  other  than  the  water  itself. 

Operating  and  dressing  rooms  in  hospitals  are  now  frequently 
provided  with  hot  and  cold  sterile  water,  which  must  be  examined 
from  time  to  time.  In  the  bacteriologic  examination  of  such  water 
the  author  has  used  the  following  method  in  order  to  exclude  the 
possibility  of  contamination  from  outside  sources.  A  150  c.c.  beaker 
is  filled  with  95  per  cent,  carbolic  acid  and  held  so  that  the  mouth 
of  the  faucet  dips  into  the  acid.  After  the  latter  has  acted  for  several 
minutes  the  beaker  is  removed  and  replaced  by  one  containing  hot 
sterile  water.  This  removes  the  carbolic  acid.  The  faucet  is  next 
opened  and  the  water  allowed  to  flow  for  several  minutes.  A  sterile 
flask  is  then  partially  filled  and  immediately  closed  with  a  cotton  plug. 

When  water  has  to  be  collected  from  a  river,  a  large  water  tank,  or 
a  pond,  it  is  desirable  to  obtain  the  specimen  at  some  distance  from 
the  shore  or  wall  of  the  tank.  This  can  be  accomplished  by  fastening 
a  string  to  a  sterile  flask  containing  lead  shot,  and  throwing  the 
vessel  out  into  the  water.  When  filled  it  is  rapidly  withdrawn,  and 
after  a  little  water  has  been  poured  out  the  flask  is  closed  with  a 
sterile  cotton  plug.  For  obtaining  water  from  a  particular  depth  of  a 
body  of  water,  special  apparatuses  have  been  constructed  by  Esmarch 
and  by  Roux.  Esmarch's  device  consists  of  a  bottle  with  a  rubber 
cap  and  weight.  It  is  lowered  on  a  line  to  a  definite  depth,  the 
rubber  cap  is  opened  by  a  string  attached  to  it,  the  bottle  is  filled, 
the  rubber  cap  closed  again  automatically,  and  the  apparatus  is 
pulled  up  to  the  surface. 


BACTERIOLOGIC  EXAMINATION  OF  SOIL  181 

Roux  uses  flasks  which  are  drawn  out  at  the  neck  into  a  thin 
twisted  capillary  glass  tube.  After  sterilization  they  should  contain 
a  small  amount  of  distilled  water,  which  is  heated  over  an  open  flame 
to  boiling  and  evaporated  down  to  a  small  residue.  The  capillary 
tube  is  then  fused  over  a  flame  and  the  bottles  cooled,  when  they  will 
be  found  to  contain  a  vacuum.  A  heavy  string  is  attached  to  the 
closed  capillary  tube  and  the  bottle  enclosed  in  a  metal  capsule, 
which  is  lowered  into  the  water.  When  the  apparatus  is  at  the  desired 
depth  the  string  is  pulled,  breaking  the  capillary  tube  and  allowing 
the  water  to  rush  into  the  vacuum  in  the  flask.  The  apparatus  is 
then  drawn  to  the  surface. 

A  trustworthy  bacteriologic  examination  of  water  can  only  be  made 
if  the  plates  are  prepared  on  the  spot  where  the  samples  are  collected. 
If  water  is  removed  to  a  distance,  even  when  packed  in  ice,  the 
subsequent  count  of  the  colonies  does  not  furnish  an  accurate  result, 
because  some  water  bacteria  multiply  near  the  freezing  point,  while 
others  are  killed  by  chilling. 

Inoculation  of  Culture  Media. — The  culture  media  must  be  inocu- 
lated with  definite  amounts  of  the  water.  For  this  purpose  1  c.c. 
pipettes,  subdivided  into  0.1  and  0.01  c.c.,  are  required.  These 
pipettes  should  be  placed  in  glass  tubes  which  are  fused  at  one  end 
and  closed  with  a  cotton  plug  at  the  other.  The  upper  ends  of  the 
pipettes  themselves  must  also  be  closed  with  cotton  plugs,  and  they 
and  the  tubes  sterilized  in  the  hot-air  sterilizer.  Water  containing 
few  bacteria  may  be  mixed  with  the  culture  media  in  quantities  of 
between  1.0  and  0.1  c.c.  When  there  are  many  bacteria,  as  in  the 
case  with  water  contaminated  by  sewage,  the  sample  must  be  diluted 
with  sterile  distilled  water  before  being  mixed  with  the  culture  media. 
The  sterile  water  is  brought  to  the  place  of  examination  in  volumetric 
flasks  in  quantities  of  25  c.c.,  50  c.c.,  100  c.c.,  and  250  c.c.  One  c.c. 
of  the  suspected  water  is  added  to  the  sterile  distilled  water  in  the 
volumetric  flask.  The  latter  is  well  shaken  and  1  c.c.  of  the  diluted 
sample  is  added  to  the  culture  medium  with  a  fresh  sterile  pipette. 
The  culture  media  used  for  the  bacterial  examination  of  water  should 
be  of  a  very  definite  degree  of  alkalinity.  It  is  necessary  to 
use  both  gelatin  and  agar  plates.  They  should  be  kept  at  a  tempera- 
ture of  20°  C.  for  eight  days,  since  many  water  bacteria  grow  slowly 
on  artificial  culture  media.  If  water  is  examined  with  special 
reference  to  certain  pathogenic  bacteria,  special  culture  media  and 
incubator  temperature  are  necessary.  The  most  important  pathogenic 
bacteria  occurring  in  water  are  the  typhoid  bacillus  and  the  cholera 
spirillum. 

Bacteriologic  Examination  of  Milk. — The  bacteriology  of  milk  will  be 
treated  in  subsequent  chapters. 

Bacteriologic  Examination  of  Soil. — Qualitative  Examination. — As 
already  stated  this  is  chiefly  undertaken  for  the  detection  of  patho- 
genic bacteria  like  those  of  anthrax,  tetanus,  and  malignant  edema. 


182     EXAMINING  AIR,  SOIL,  WATER,  AND  FLUIDS  FOR  BACTERIA 

These  microorganisms  are  most  readily  detected  by  direct  animal 
inoculations.  The  search  for  nitrifying  and  other  bacteria,  important 
in  the  study  of  agricultural  problems,  requires  certain  special  culture 
media  and  the  dilution  of  the  finely  divided  soil  with  sterile  distilled 
water. 

Quantitative  Examination. — This  is  very  unreliable  and  generally 
not  very  satisfactory.  In  the  first  place  it  is  difficult  to  divide  the  soil 
so  finely  that  it  can  be  intimately  mixed  with  melted  culture  media, 
and,  again,  the  soil  bacteria  (mostly  bacilli)  have  such  a  wide  range  of 
conditions  of  growth  that  no  single  cultural  method  can  furnish  even 
an  approximate  picture  of  the  species  and  number  of  germs  present 
in  a  specimen  of  soil.  For  example,  there  are  aerobic  and  anaerobic 
bacteria,  some  growing  at  very  low  and  others  at  extremely  high 
temperatures. 

Specimens  of  soil  from  the  superficial  strata  are  generally  obtained 
with  a  small  platinum  spoon  or  scoop  containing,  when  filled,  about 
5*0  or  2*5-  of  a  cubic  centimeter.  The  material  may  first  be  rubbed 
up  in  a  sterile  agate  mortar,  or  it  may  be  mixed  directly  by  violent 
snaking  with  the  melted  culture  medium,  which  is  subsequently 
poured  into  Petri  dishes.  For  the  examination  of  soil  of  deeper  strata 
a  drill  or  auger  has  been  devised  by  Frankel.  Above  the  tip  is  a 
metal  tube  with  an  opening  on  one  side,  with  a  cover  arrangement 
on  hinges  like  a  door.  During  the  downward  movement  the  door 
remains  closed,  but  as  soon  as  the  motion  is  reversed  it  opens  and 
the  tube  is  filled  with  soil.  In  this  manner  it  is  possible  to  obtain  soil 
from  a  definite  depth. 

FIG.  107 


Tip  of  FrankePs  instrument  for  obtaining  earth  from  various  depths  for  bacteriologic  study. 
B  shows  the  instrument  with  its  cavity  closed,  as  it  appears  during  boring:  A,  open,  as  it 
appears  when  twisted  in  the  other  direction  to  collect  the  earth.  (McFarland.) 

Bacteriologic  examination  has  shown  that  soils  are  generally  richer 
in  bacteria  the  more  they  have  been  mixed  or  contaminated  with 
manure,  sewage,  and  other  decomposing  vegetable  or  animal  material ; 
further,  that  this  richness  is  confined  to  the  superficial  layers.  At  a 
depth  of  1  meter  the  number  of  bacteria  is  much  reduced,  and  at 
a  depth  of  1J  to  2  meters  very  few  are  present;  still  deeper  there  are 
none  at  all. 


QUESTIONS  183 


QUESTIONS. 

1.  What  is  the  general  principle  underlying  the  bacteriologic  examination  of 
soil,  water,  milk? 

2.  What  is  the  principle  on  which  the  method  for  quantitative  bacteriologic 
air  examination  is  based  ? 

3.  What  is  meant  by  a  quantitative,  by  a  qualitative  bacteriologic  exami- 
nation of  soil,  water,  air,  etc.? 

4.  What  method  is  used  when  soil  is  to  be  examined  for  the   presence  of 
tetanus  bacilli  ? 

5.  How  can  air  be  examined  for  the  presence  of  tubercle  bacilli? 

6.  Describe  the  arrangement  and  use  of  a  W^olffhiigel  counting  apparatus. 

7.  What  is  a  square  centimeter;  what  a  square  millimeter?    What  is  a  cubic 
centimeter  ? 

8.  How  many  colonies  are  in  the  culture  medium  of  a  Petri  dish  8  cm.  in 
diameter  if  on  an  average  each  square  centimeter  contains  twenty-three  colonies? 

9.  What  is  an  Esmarcjh  roll-tube? 

10.  Describe  a  simple  method  of  approximately  estimating  the  number  of 
germs  in  air  by  the  aid  of  exposed  Petri  dishes. 

11.  Describe  another  method  by  the  aid  of  large  Erlenmeyer  flasks  filled  with 
water. 

12.  What  is  a  liter?    What  is  its  equivalent  in  pints? 

13.  Describe  Hesse's  method  of  quantitative  bacteriologic  air  examination. 

14.  Describe  Eyre's  method  of  aspirating  air  for  a  quantitative  bacteriologic 
analysis. 

15.  Describe  the  method  of 'using  sand  or  sugar  as  filters  for  catching  the 
bacteria  in  aspirated  air. 

16.  What  is  the  Sedgwick-Tucker  modification  of  the  air-filtering  tube? 

17.  How  are  flasks  used  in  collecting  water  for  quantitative  bacterial  analysis 
to  be  sterilized  and  handled  before  use  ? 

18.  How  can  water  running  from  a  faucet  be  prevented  from  washing  down 
bacteria  from  the  latter  into  the  collecting  flask  ? 

19.  How  is  water  collected  from  a  river  or  pond? 

20.  What  devices  are  used  for  collecting  water  from  a  particular  depth? 

21.  Describe  the  method  of  obtaining  definite  amounts  of  water  from  the 
collecting  bottles  for  the  inoculation  of  culture  media. 

22.  WTiat  procedure  is  employed  with  a  specimen  of  water  containing  many 
bacteria  in  order  to  avoid  getting  an  uncountable  number  of  colonies  in  the 
Petri  dishes? 

23.  At  what  temperature  and  how  long  shall  the  Petri  dishes  be  kept  before 
the  count  of  the  colonies  is  undertaken  ? 

24.  Why  do  quantitative  bacterial  soil  examinations  furnish  very  unreliable 
figures  ? 

25.  Describe  the  method  of  collecting  soil  from  the  surface.    Also  method  of 
obtaining  soil  from  any  desired  depth. 


CHAPTEE    XV. 

PRINCIPLES  OF  DISINFECTION— DISINFECTANTS. 

WHEREVER  there  is  disease  due  to  pathogenic  microorganisms  the 
latter  will,  through  exhalation,  secretions,  and  excretions,  direct  or 
indirect  contact,  transport  by  insects  or  otherwise,  soil  or  contaminate 
objects  in  the  neighborhood  of  the  sick  animal.  When  stables,  barns, 
harness,  feed,  water  supply,  or  any  other  objects  are  soiled  or  con- 
taminated with  pathogenic  bacteria  they  are  said  to  be  infected.  The 
removal  or  destruction  of  such  infecting  bacteria  is  called  disin- 
fection. Disinfection  may  sometimes  take  place  without  destruction 
of  the  bacteria,  as  in  the  filtration  of  water  through  filters  so  dense  as 
to  prevent  the  passage  of  the  microorganisms.  As  a  rule,  however, 
the  object  of  disinfection  is  to  destroy  the  pathogenic  bacteria. 
Physical  and  chemical  means  may  be  employed  for  this  purpose. 
Among  the  former,  heat  and  sunlight  are  particularly  important; 
among  the  latter  such  chemicals  as  accomplish  the  object  in  com- 
paratively weak  concentrations. 

Effectiveness  of  Disinfectants. — The  effect  of  every  disinfectant  must 
be  studied  separately  for  each  type  of  pathogenic  organism  and  also 
for  the  vegetative  forms  and  spores  of  sporulating  bacteria.  The 
spores  are,  as  has  been  previously  pointed  out,  much  more  resistant 
to  all  disinfecting  procedures  than  the  vegetative  forms.  In  the 
theoretical  laboratory  study  of  disinfection  it  is  necessary  to  ascertain 
not  merely  the  circumstances  under  which  physical  or  chemical 
means  kill  all  bacteria,  but  also  to  find  out  at  what  degree  or  con- 
centration they  have  a  marked  hindering  or  inhibiting  influence  upon 
bacterial  growth  and  multiplication.  In  practice  the  absolute  killing 
of  all  infecting  bacteria  is  seldom  possible.  Diluting,  diminishing, 
and  damaging  them  in  such  a  way  that  their  growth  is  inhibited  and 
they  are  robbed  of  much  of  their  virulency  must  generally  be  con- 
sidered as  sufficient.  The  simplest  and  most  potent  physical  agent 
used  for  disinfection  is  heat.  Bacteria  grow  best  at  a  certain  temper- 
ature, called  their  optimum  temperature;  any  degree  above  it  retards 
their  growth,  and  any  degree  above  their  maximum  temperature 
inhibits  growth  entirely  and  generally  damages  them  seriously.  When 
working  with  chemical  agents  the  least  concentration  which  will 
entirely  inhibit  growth  must  first  be  ascertained,  and  then  the  con- 
centration which  will  destroy  bacteria  and  the  necessary  time  to 
accomplish  it.  Absolutely  harmless,  non-pathogenic  bacteria,  some 
of  which  form  spores  more  resistant  than  the  spores  of  any  pathogenic 


TESTING  THE  EFFECT  OF  TEMPERATURE  185 

bacteria  are  not  considered  in  disinfection.  The  destruction  of  all 
life  in  a  medium,  as  has  already  been  explained,  is  called  sterilization; 
disinfection,  however,  does  not  go  to  this  extent. 

The  determination  of  the  exact  disinfecting  value  of  either  physi- 
cal or  chemical  agents  is  more  difficult  than  would  appear  on  first 
sight.  It  is  always  necessary  in  such  experiments  to  use  bacteria 
which  have  been  grown  under  the  most  favorable  conditions  and 
which  are  derived  from  a  young,  vigorous  culture.  Another  important 
factor  is  whether  these  bacteria  are  contained  in  distilled  water,  salt 
solution,  or  a  favorable  fluid  culture  medium,  because  in  the  latter 
they  are  generally  more  resistant  than  in  pure  water.  It  is  also 
necessary  to  protect  them  from  any  damaging  influences  before  the 
actual  test. 

Theobald  Smith  has  shown  that  tetanus  spores  obtained  under  the 
most  favorable  conditions  can  resist  moist  heat  for  over  one  hour, 
while  those  raised  in  media  containing  sugar  and  previously  damaged 
by  the  acid  which  they  have  formed  from  it  are  much  less  resistant. 

The  action  of  the  disinfectant  is  also  much  influenced  by  the 
presence  of  certain  substances  in  the  culture  medium.  Corrosive 
sublimate,  for  example,  is  much  weakened  in  the  presence  of  albumin- 
oid or  proteid  bodies  or  discharges  containing  them.  After  the  test 
the  removal  of  the  disinfectant  from  the  bacteria  by  washing  or 
chemical  means  is  another  important  factor. 

Testing  the  Effect  of  Temperature. — The  method  used  for  testing 
the  effect  of  temperature  upon  bacteria  is  the  following:  Culture 
tubes  containing  a  few  cubic  centimeters  of  young  bouillon  cultures 
of  the  organism  are  removed  from  the  incubator  and  placed  in  a 
water  bath  containing  a  large  amount  of  water  heated  to  a  certain 
stationary  temperature.  One  of  the  tubes  must  contain  a  thermometer 
in  order  to  indicate  the  exact  moment  when  the  temperature  in  the 
culture  medium  coincides  with  that  of  the  water  bath.  The  tubes  are 
then  heated  for  varying  periods  of  time — for  example,  for  five,  seven, 
ten,  eleven,  and  twelve  minutes,  and  so  on.  They  are  taken  out  of 
the  water  bath  one  by  one  and  at  once  plunged  into  cold  water. 
Upon  the  conclusion  of  this  process,  culture  tubes  with  liquefied  solid 
media  are  inoculated,  plates  are  poured,  and  the  developing  colonies 
are  subsequently  counted.  The  first  plate  remaining  sterile  indicates 
the  time  exposure  necessary  to  kill  the  organism  at  the  temperature 
used  in  the  experiment.  The  author  has  found  this  method  inaccurate 
by  furnishing  values  which  are  too  low,  because  the  small  quantity 
of  the  original  cultures  transferred  in  pouring  the  Petri  dishes  may 
not  contain  any  live  bacteria,  though  an  appreciable  number  of 
especially  resistant  individual  organisms  may  be  present  in  the  whole 
bulk  of  the  bouillon.  An  absolutely  trustworthy  method  is  the 
following  : 

The  tubes  are  heated  and  cooled  as  previously  described,  and  then 
the  entire  contents  of  each  tube  (for  example,  5  or  10  c.c.),  after 


186  PRINCIPLES  OF  DISINFECTION 

flaming  the  mouth,  is  poured  into  a  flask  containing  100  c.c.  of 
sterile  bouillon.  The  flasks  are  incubated  for  forty-eight  hours  and 
solid  media  are  inoculated  from  these.  By  this  method  even  a  single 
surviving  bacterium  is  given  an  opportunity  for  subsequent  develop- 
ment. The  effect  of  sunlight  and  other  physical  influences  may  be 
ascertained  in  an  identical  manner. 

Testing  the  Effect  of  Chemical  Disinfectants  or  Antiseptics. — The 
method  generally  employed  is  the  following:  Sterile  silk  threads  are 
soaked  in  culture  media  containing  the  bacteria  whose  resistance  is 
to  be  tested.  The  threads  are  afterward  dried  and  suspended  for 
different  periods  of  time  in  solutions  of  varying  strength  of  the  anti- 
septic. The  latter  is  then  washed  off  in  sterile  distilled  water  and 
the  silk  threads  are  immersed  in  fluid  or  liquefied  solid  culture  media. 
Since  there  are  certain  objectionable  features  in  the  silk-thread 
method  it  has  been  modified,  and  small  sterile  garnet  crystals  on 
which  the  bacteria  are  dried  are  used  to  replace  the  threads.  The 
crystals  are  treated  in  the  same  manner  as  the  threads.  All  chemical 
disinfectants  act  more  quickly  at  high  than  at  low  temperatures;  that 
is  at  high  temperatures  which  are  not  in  themselves  damaging.  This 
observation  corresponds  with  the  general  law  that  chemical  reactions 
take  place  more  promptly  and  more  rapidly  at  higher  temperatures. 

Dry  and  Moist  Heat. — One  of  the  most  important  considerations  in 
the  study  of  heat  in  disinfection  is  the  fact  that  dry  heat  is  very 
much  less  effective  than  moist  heat.  This  is  particularly  apparent 
in  the  case  of  spores.  Anthrax  spores,  for  instance,  can  withstand 
dry  heat  at  100°  to  120°  C.  for  several  hours,  and  even  at  140°  C.  their 
absolute  destruction  is  only  accomplished  after  three  hours,  while 
water  boiling  at  100°  C.  kills  them,  even  at  the  highest  estimate,  in 
twelve  minutes.  The  effect  of  steam  developed  under  pressure  in 
the  autoclave  is  still  more  powerful.  The  spores  of  certain  soil  and 
potato  bacilli  can  withstand  streaming  steam  at  100°  C.  for  over 
sixteen  hours,  but  they  are  killed  in  steam  under  pressure  at  105°  to 
110°  C.  in  from  five  to  fifteen  minutes  and  at  140°  C.  in  one  minute. 
Steam  developed  under  reduced  pressure  at  a  lower  temperature,  as 
at  high  altitudes  or  in  experimental  work  is,  of  course,  less  effective 
in  killing  spores  than  steam  at  the  ordinary  pressure  of  one  atmos- 
phere and  at  100°  C.  Overheated  steam  not  saturating  the  atmos- 
phere in  which  the  spores  are  exposed  also  has  a  reduced  destructive 
power.  Esmarch  and  Kokubo  found  that  a  very  small  admixture  of 
antiseptics,  (creosote,  formalin,  etc.)  to  the  streaming  steam,  enor- 
mously increased  its  destructive  power  toward  spores.  Dry  heat  is 
not  only  much  less  powerful  as  a  disinfectant  than  moist  heat,  but  is 
also  relatively  of  much  less  value  because  it  has  little  penetrating 
power.  Koch  and  Wolffhiigel  showed  experimentally  that  in  a  bale 
composed  of  nineteen  woollen  blankets,  which  had  been  exposed  for 
three  hours  to  dry  heat  of  130°  to  140°  C.,  the  temperature  in  the 
interior  had  only  risen  to  35°  C. 


THE  IDEAL  DISINFECTANT  187 

Cold.— Cold  has  practically  no  effect  upon  bacteria  and  their 
spores;  alternately  freezing  at  very  low  temperatures  and  subsequent 
thawing,  however,  destroys  the  vegetative  form  of  some  pathogenic 
bacteria.  Cold  has,  accordingly,  no  place  in  the  armamentarium  of 
practical  disinfection. 

Direct  Sunlight. — The  chemically  active  blue,  violet,  and  ultra- 
violet rays  of  the  spectrum  have  a  particularly  strong  germicidal  effect 
upon  both  the  vegetative  forms  and  spores.  The  effect,  however,  is 
limited  to  the  surface  of  objects.  The  beneficial  effect  of  sunlight 
should  not  be  underestimated,  particularly  in  veterinary  practice, 
where  the  great  majority  has  not  yet  learned  the  importance  of  having 
stables,  barns,  and  other  places  where  animals  are  kept  well  lighted 
during  the  day  and  particularly  well  lighted  and  exposed  to  the  sun 
after  the  prevalence  of  infectious  diseases.  Arloing  noted  the  effect 
of  sunlight  upon  anthrax  bacilli  and  spores.  Pansini  showed  that  the 
bacilli  in  cultures  are  killed  in  from  one  to  two  and  one-half  hours; 
the  moist  spore  directly  exposed  in  one-half  to  two  hours;  the  dried 
spores  in  from  six  to  eight  hours.  Koch  and  others  demonstrated 
the  germicidal  effect  of  sunlight  upon  tubercle  bacilli.  Rosenau 
found  that  the  plague  bacillus  exposed  to  the  direct  action  of  the  sun- 
light dies  in  half  an  hour,  provided  that  the  temperature  is  above  30°  C. 
Even  diffuse  daylight,  when  acting  long  enough,  has  an  inhibiting 
and  detrimental  effect  upon  many  pathogenic  bacteria,  but  it  is  very 
much  slower  than  the  effect  of  direct  insolation. 

Drying. — Some  bacteria,  like  the  glanders  bacillus,  the  cholera 
spirillum,  etc.,  are  very  rapidly  killed  by  drying  out,  while  others,  like 
the  tubercle  bacillus,  are  only  affected  after  they  have  been  dried  out 
for  a  long  time.  Certain  spores,  like  those  of  anthrax,  remain  alive 
and  virulent  even  after  many  years. 

Electric  Currents,  X-rays,  and  Radium. — Electric  currents,  accord- 
ing to  Zeit  and  others,  have  no  direct  effect  upon  bacteria,  unless 
they  are  in  solutions  which  give  rise  to  detrimental  electrolytic  prod- 
ucts, such  as  acids,  ozone,  etc.  The  arrays,  according  to  the  same 
author,  appear  to  have  no  germicidal  effect.  Radium  emanations 
have  an  appreciable  effect;  they  have  killed  anthrax  spores  after  an 
exposure  of  three  days.  This  effect,  however,  seems  to  depend  entirely 
upon  the  easily  absorbable  rays  and  not  upon  the  deeply  penetrating 
rays,  and  is,  therefore,  only  superficial. 

The  "Ideal"  Disinfectant. — An  ideal  disinfectant  would  be  one 
which,  while  highly  efficient,  deeply  penetrating,  and  absolutely  trust- 
worthy, would  not  damage  any  of  the  objects  to  be  disinfected.  Such 
a  disinfectant  does  not  exist.  Moist  heat  is  excellent  for  cotton,  wool, 
and  linen,  but  it  destroys  leather,  harness,  etc.  Formalin  is  strong 
and  effective,  does  not  easily  damage,  but  its  penetrating  power  is 
deficient.  Some  of  the  most  important  disinfectants  extensively  used 
in  practical  every-day  work  are  the  following: 


188  PRINCIPLES  OF  DISINFECTION 

Corrosive  Sublimate,  or  Bichlorid  of  Mercury  (HgCl2). — This  is  one 
of  the  strongest  antiseptics  known,  but  its  effectiveness  is  much  de- 
creased in  the  presence  of  albuminoids.  This  disadvantage  can  be 
partially  corrected  by  adding  hydrochloric  acid,  tartaric  acid,  citric 
acid,  chloride  of  sodium,  and  other  chemicals  to  the  corrosive  sublimate 
solutions.  Bichlorid  solutions  are  also  objectionable  in  that  they 
corrode  metals,  and  are  very  poisonous.  There  is,  however,  hardly 
any  better  disinfectant  for  stables  and  barns  which  have  been  infected 
with  anthrax  bacilli  and  their  spores.  In  this  case  the  disinfectant 
should  be  used  in  the  strength  of  1  to  500,  and  it  is,  of  course,  necessary 
to  proceed  systematically  so  that  all  surfaces  of  walls  and  floors  which 
may  have  become  contaminated  are  exposed  to  the  action  of  the 
solution.  This  may  be  accomplished  by  the  use  of  mops  or  brooms 
or  an  elevated  tank  connected  with  a  rubber  hose,  or  best  by  the 
employment  of  a  pressure  pump.  After  thoroughly  wetting  every 
surface  the  solution  should  be  allowed  to  act  for  from  one  to  two  hours, 
and  then  the  place  should  be  well  cleaned  with  water.  In  the  use 
of  bichlorid  solutions  it  must  be  remembered  that  they  are  very  power- 
ful poisons  to  man  and  domestic  animals,  and  that  subsequent  removal 
by  very  thorough  washing  and  flushing  is  necessary.  Bichlorid  can- 
not be  used  to  disinfect  tubercular  material,  such  as  sputum,  caseous 
masses,  etc.,  because  the  mercury  salt  forms  a  compound  with  albu- 
minoids of  very  slight,  if  any,  disinfectant  value. 

Caustic  Lime. — Among  other  salts  of  the  metals  much  used  in 
practical  disinfection,  caustic  lime  deserves  to  be  mentioned.  Its 
effect  depends  upon  its  strongly  alkaline  reaction.  On  this  account 
old  solutions  in  which  the  lime  salt  has  combined  with  carbon  dioxide 
and  formed  carbonate  of  lime  are  of  little  or  no  value.  The  solution 
must,  therefore,  be  prepared  immediately  before  use.  The  following 
formula  is  recommended.  Take  a  number  of  pounds  of  pure  burnt  lime 
(CaO),  add  slowly  and  gradually  for  each  ten  pounds  1^  gallons  of 
water,  and  finally  add  20  gallons  of  water  and  mix  well.  This  mix- 
ture will  represent  a  20  per  cent,  milk  of  lime  which  when  fresh  is 
quite  an  effective  disinfectant. 

Chlorinated  Lime. — This  is  also  known  as  chloride  of  lime,  or  bleach- 
ing powder,  and  is  prepared  by  passing  a  stream  of  nascent  chlorine 
gas  over  moist  unslaked  lime  (calcium  hydrate).  It  is  a  soft  white 
friable  substance,  very  slightly  soluble  in  water,  and  of  indefinite 
chemical  composition.  Its  disinfecting  value  depends  chiefly  upon 
the  amount  of  calcium  hypochloride  which  it  contains.  Its  bleaching 
and  destructive  properties  limit  its  employment  as  a  germicide,  but  it  is 
used  extensively  in  stables  and  barns  for  the  disinfection  of  infected 
fecal  matter,  urine,  manure,  bedding,  floors,  and  woodwork,  etc. 

Permanganate  of  Potash. — In  a  4  per  cent,  solution  this  is  a  very 
powerful  disinfectant  and  kills  anthrax  spores  in  about  fifteen  minutes. 

Carbolic  Acid. — Carbolic  acid  (C6H5OH)  and  other  bodies  belonging 
to  this  group  of  chemicals  are  very  important  disinfectants.  They 


FORMALDEHYDE  189 

are  not  as  effective  as  corrosive  sublimate,  and  have  to  be  used  in 
much  stronger  solutions  when  they  are  very  poisonous;  but  they  are 
quite  penetrating,  and  their  value  is  not  decreased  by  the  presence 
of  albuminoid  bodies,  which  gives  them  a  wide  range  of  application. 
Carbolic  acid  is  generally  used  in  3  to  5  per  cent,  solutions.  Its 
effect  is  very  much  increased  by  warming  it  to  about  40°  C.,  or  by 
the  addition  of  hydrochloric  acid,  but  not  by  the  addition  of  alcohol. 
Other  bodies  of  the  carbolic-acid  group  used  as  disinfectants  are  the 
creosotes  (ortho-,  meta-,  and  para-creosote,  the  mixture  known  as 
tri-creosote),  creolin,  and  lysol.  For  the  cleansing  of  woodwork,  floors, 
w^alls,  etc.,  Nocht's  carbol-soap  solution  is  highly  recommended.  It  is 
prepared  as  follows :  Dissolve  6  per  cent,  soft  soap  in  hot  water  and 
add  to  the  hot  solution  5  per  cent,  of  raw  (100  per  cent.)  carbolic 
acid.  Should  any  drops  of  tar  form  they  should  be  removed  and 
only  the  clear  solution  used.  It  does  not  stain  and  cleanses  wood- 
work thoroughly. 

Formaldehyde. — Formaldehyde  is  the  aldehyde1  of  methylic  alcohol. 
It  is  a  gaseous  body  of  the  chemical  formula  CHOH.  It  is  found  in 
commerce  in  the  form  of  a  watery  solution,  which  should  contain 
forty  volumes  of  the  gas  per  one  volume  of  water.  This  solution  in 
addition  to  being  simply  called  formaldehyde  is  also  known  under 
a  variety  of  proprietary  names,  such  as  formol,  formalin,  etc.  When  a 
solution  of  formaldehyde  is  warmed,  and  often  merely  upon  standing 
undisturbed,  a  part  of  the  gaseous  body  becomes  polymerized,  which 
means  that  several  molecules  unite  to  form  a  larger  molecule.  Gen- 
erally three  molecules  of  formaldehyde  unite  and  form  a  polymer- 
ization product  with  the  formula  (H — CHO)3.  This  body  is  known 
as  trioxymethylene,  para-formaldehyde,  or  simply  as  paraform.  It  is  in- 
soluble in  water,  and  forms  a  white  sediment  in  the  vessel  containing 
the  formaldehyde  solution.  This  chemical  change  is  important, 
because  such  a  white  precipitate  or  sediment  indicates  that  a  formalin 
solution  has  lost  much  of  its  formaldehyde,  that  its  value  as  a  fluid 
or  gaseous  disinfectant  has  become  weakened,  and  that  it  must  be 
used  afterward  with  a  proper  knowledge  and  consideration  of  this 
change.  In  making  up  formalin  solutions  it  must  always  be  remem- 
bered that  the  best  commercial  product  contains  not  more  than  40  per 
cent,  of  the  disinfectant  itself.  Hence,  if  a  4  per  cent,  formalin  solu- 
tion is  recommended  for  a  certain  procedure  it  must  be  prepared 
by  taking  one  part  of  formalin  and  nine  parts  of  water,  not  one  part 
of  formalin  and  twenty-five  parts  of  water.  Paraform  is  also  used 
as  a  disinfectant,  but  according  to  a  different  method  from  that 
employed  with  the  gaseous  formaldehyde  dissolved  in  water.  The 
germicidal  effect  of  a  4  per  cent,  formalin  solution  used  as  a 
disinfectant  is  about  equivalent  to  a  1  to  1000  solution  of  corrosive 

1  An  aldehyde  is  a  body  formed  by  the  oxidation  of  a  primary  or  secondary  alcohol,  that  is, 
by  the  substitution  of  two  or  one  H  atom  in  the  alcohol  by  one  O  atom. 


190 


PRINCIPLES  OF  DISINFECTION 


sublimate  and  to  a  5  per  cent,  solution  of  carbolic  acid.  The 
antiseptic  value  of  formalin  is  very  high,  and  it  will  inhibit  the 
growth  of  bacteria  if  present  in  a  proportion  of  1  to  25  to  50,000; 
for  germicidal  purposes  a  J  per  cent  solution  should  be  employed. 

When  formaldehyde  was  first  introduced  as  a  disinfectant  it  was 
used  by  allowing  the  gas  to  evaporate  unaided  from  the  watery 
solution.  This  is  a  slow  process,  requiring  long  exposures  and  large 
amounts  of  formalin  to  be  in  any  way  effective.  Later,  Trillat  devised 
a  method,  using  a  lamp  of  his  own  construction,  in  which  methyl- 
alcohol  was  evaporated  under  conditions  that  oxidized  it  into  formal- 
dehyde; but  since  the  yield  was  only  7  to  8  per  cent,  of  the  methyl- 
alcohol  used,  this  process  was  expensive,  slow,  and  not  very  efficient. 
Other  arrangements  allow  the  evaporation  of  formalin  with  such 
additions  (20  per  cent,  chloride  of  calcium-glycerin)  that  the  forma- 
tion of  paraform  is  prevented.  Still  other  devices  are  based  upon 
the  heating  and  decomposition  of  paraform  into  formaldehyde. 

Fio.  108 


Breslau  regenerator  and  lamp. 

According  to  Gotschlich,  the  best  formaldehyde  disinfecting  pro- 
cedure is  that  of  Fliigge  and  his  assistants.  It  is  known  as  the  Breslau 
method,  and  consists  in  evaporating  a  dilute  formalin  (1  part  of  for- 
malin to  4  parts  of  water)  in  a  simple  apparatus.  It  has  been  found 
that  such  dilute  formalin  solutions  do  not  upon  evaporation  form 
paraform,  but  allow  all  the  formaldehyde  present  in  solution  to  be 
expelled  with  the  evaporating  water.  Harrington  recommends  very 
highly  a  formalin  disinfecting  apparatus  devised  by  Professor  Robin- 
son, of  Bowdoin  College;  also  a  regenerator  made  by  the  Sanitary 
Construction  Company,  which  is  said  to  be  simple  and  economical. 
Paraform  evaporation  is  brought  about  by  the  Schering  paraform 
lamp. 

Before  using  any  of  the  apparatus  mentioned,  and  formaldehyde 


QUESTIONS 


191 


FIG.  109 


in  the  gaseous  state  in  any  form  for  the  disinfection  of  the  habitation 
of  man  and  domestic  animals,  all  doors,  windows,  cracks,  and  openings 
of  any  sort  should  be  closed  tight.  In  the  case  of  barns,  stables,  etc., 
this  task  is  not  always  easily  accomplished.  The  amount  of  formalin 
necessary  for  the  disinfection  of  any  space  depends  upon  its  cubical 
contents.  Approximately  a  pint  of  40  per  cent,  formalin  or  60  pastilles 
of  paraform  must  be  evaporated  for  each  1000  cubic  feet  of  space. 
With  the  Breslau  method  about  J  pint  for 
1000  cubic  feet  is  sufficient  in  a  seven 
hours'  exposure.  Any  formaldehyde  re- 
maining at  the  termination  of  disinfection 
may  be  neutralized  by  ammonia  vapors. 
Formaldehyde  is  not  an  insecticide,  and 
has  no  effect  on  these  animals. 

Sulphur  Dioxide. — Besides  formaldehyde, 
sulphur  dioxide  is  the  only  other  substance 
which  is  still  extensively  used  as  a  gaseous 
disinfectant.  It  is  produced  by  burning 
sulphur.  The  simplest  and  best  arrange- 
ment is  to  place  flowers  of  sulphur  into 
an  iron  pot,  which  is  placed  in  a  second 
metal  vessel  containing  a  quantity  of 
water.  This  arrangement  reduces  the 
danger  of  fire  to  a  minimum,  and  by  the 
heating  and  boiling  of  the  water  which 
will  occur  the  amount  of  water  vapor  is 
supplied  necessary  to  convert  the  sulphur 
dioxide  into  sulphurous  acid,  which  is  an 
effective  germicide.  Sulphur  dioxide,  in 
addition  to  destroying  germs,  also  kills 
insects  and  vermin,  such  as  mice  and 
rats.  Five  pounds  of  sulphur  should  be 
burned  for  each  1000  cubic  feet  of  space, 
and  one  pound  of  water  should  be  vapor- 
ized for  each  five  pounds  of  sulphur.  Spaces  to  be  disinfected  by 
burning  sulphur  must,  of  course,  be  made  as  air-tight  as  possible. 

Alcohol. — It  should  be  remembered  that  absolute  alcohol  has  prac- 
tically no  germicidal  power.  There  is,  however,  considerable  germi- 
cidal  value  in  50  per  cent,  alcohol,  and  it  is  well  adapted  for  use  in 
disinfecting  surgeons'  hands  and  the  skin  of  animals  before  operations. 


Sanitary  Construction  Company's 
regenerator. 


QUESTIONS. 

1.  What  is  meant  by  infected  objects? 

2.  What  does  the  term  disinfection  signify? 

3.  Can  pathogenic  microorganisms  be  removed  from  water  without  destroying 
them,  and  how? 

4.  What  means  different  in  type  can  be  employed  for  disinfecting  purposes? 


192  PRINCIPLES  OF  DISINFECTION 

5.  How  can  the  effect  of  a  certain  degree  of  heat  upon  pathogenic  bacteria 
be  ascertained? 

6.  What  is  meant  by  an  inhibiting  degree  of  temperature  upon  a  bacterium? 
What  by  a  germicidal  effect? 

7.  What  is  the  optimum,  what  the  maximum  temperature  of  a  bacterium? 

8.  How  is  the  effect  of  chemicals  upon  bacteria,  both  their  antiseptic  and 
their  germicidal  effect,  ascertained? 

9.  What  precautions  are  necessary  to  get  trustworthy  values  for  concen- 
tration and  time? 

10.  What  factors  influence  the  effect  of  an  antiseptic  or  disinfectant  upon 
bacteria? 

11.  Describe  the  silk  thread  and  the  garnet  method  of  testing  germicidal 
values. 

12.  What  is  the  difference  between  dry  and  moist  heat  as  a  germicide? 

13.  Give  the  effect  upon  the  germicidal  value  of  moist  heat — of  temperature, 
pressure,  complete  or  partial  saturation,  admixture  with  antiseptics. 

14.  What  is  the  effect  of  low  temperatures  upon  pathogenic  bacteria? 

15.  What  is  the  effect  of  direct  sunlight  and  diffuse  light  upon  pathogenic 
bacteria? 

16.  Give  some  examples  as  to  the  effect  on  pathogenic  bacteria. 

17.  What  is  the  effect  of  diffuse  daylight? 

18.  What  is  the  effect  of  drying  out  bacteria? 

19.  What  is  the  effect  of  electrical  currents  upon  fluid  cultures  of  pathogenic 
bacteria? 

20.  What  is  the  effect  of  the  axrays? 

21.  What  is  the  effect  of  radium  emanations? 

22.  What  are  the  properties  of  an  ideal  disinfectant?    Name  a  number  of  them. 

23.  Describe  the  germicidal  properties  of  corrosive  sublimate  in  solution  under 
varying  conditions. 

24.  How  should  it  be  used  in  disinfecting  a  barn  infected  with  anthrax  bacilli 
and  spores? 

25.  Describe  the  use  of  caustic  lime  as  a  disinfectant. 

26.  Describe  the  use  of  chlorinated  lime  as  a  disinfectant.    When  is  it  used  in 
particular? 

27.  What  is  the  effect  of  a  4  per  cent,  solution  of  permanganate  of  potash  upon 
anthrax  spores? 

28.  Give  advantages  and  disadvantages  of  carbolic  acid  as  a  disinfectant. 

29.  What  is  an  aldehyde?    What  is  formaldehyde? 

30.  What  is  formol  or  formalin? 

31.  How  is  a  1  per  cent,  solution  of  formaldehyde  prepared  from  a  good  com- 
mercial formalin? 

32.  What  is  meant  by  polymerization?    What  is  the  polymerization  product 
of  formalin?    Is  it  a  gas  like  the  latter? 

33.  How  can  paraform  be  used  as  a  disinfectant? 

34.  How  does  a  4  per  cent,  solution  of  formalin  compare  with  corrosive  sub- 
limate and  with  carbolic  acid? 

35.  What  kind  of  an  acid  is  carbolic  acid? 

36.  How  should  formalin  be  used  as  a  disinfectant?    How  much  for  each  1000 
cubic  feet  of  space? 

37.  What  is  the  Breslau  method  of  using  formalin? 

38.  How  is  a  space  to  be  disinfected  by  formalin  prepared? 

39.  How  can  the  unpleasant  pungent  irritating  smell  of  formalin  be  removed 
after  disinfecting  a  space  with  it? 

40.  Discuss  the  value,  advantages,  and  disadvantages  of  sulphur  dioxide  as  a 
disinfectant. 

41.  How  is  it  prepared?    How  much  is  required  for  each  1000  cubic  feet  of 
space? 

42.  Discuss  the  value  of  100  per  cent,  and  of  50  per  cent,  alcohol  as  a  disin- 
fectant. 

43.  What  is  the  difference  between  disinfection  and  sterilization? 


PART    II. 
SPECIAL  BACTERIOLOGY. 


CHAPTEE    XVI. 

WOUND  INFECTION,  SUPPURATION,  AND  THE  COMMON 
PYOGENIC  BACTERIA. 

Pyogenic  Bacteria. — A  large  number  of  disease-producing  bacteria, 
after  having  entered  and  multiplied  in  the  body  of  man  and  the 
lower  animals,  may,  under  favorable  conditions,  lead  to  inflammatory 
reaction  with  the  formation  of  pus.  A  limited  number,  however,  are 
so  frequently  found  as  the  cause  of  and  associated  with  suppurative 
processes  that  they  are  called  the  pyogenic  bacteria.  The  word 
pyogenic  means  pus-producing.  The  pyogenic  staphylococci  and 
streptococci  are  the  most  common  of  these.  The  former,  particularly, 
are  practically  ubiquitous;  they  are  found  in  the  air,  soil,  water,  and 
on  the  external  and  internal  gastro-intestinal  surfaces  of  man  and 
animals.  In  the  external  world  they  ordinarily  exist  as  saprophytes, 
and,  as  a  rule,  cannot  enter  the  body  through  a  healthy  skin  or  mucous 
membrane.  Absolutely  clean  wounds,  such  as  are  made  by  the  sur- 
geon with  every  possible  aseptic  precaution,  will  heal,  as  it  is  termed, 
by  primary  (first)  intention,  and  will  not  suppurate.  Wounds,  how- 
ever, which  are  received  under  natural  conditions,  will  suppurate 
unless  they  are  immediately  cleansed  with  antiseptic  solutions  and 
dressed  to  exclude  the  air  and  other  possible  sources  of  contamination. 
The  suppuration  may  vary  in  extent  from  being  so  scanty  that  it 
can  only  be  recognized  microscopically  to  being  so  great  that  there 
is  a  constant  abundant  discharge.  The  variations  are  dependent  upon 
the  species  of  the  infecting  pyogenic  organism  and  upon  the  greater 
or  lesser  susceptibility  of  the  infected  race  or  individual.  It  is  well 
known  that  certain  species  of  animals  are  very  susceptible  to  certain 
pyogenic  organisms  while  others  are  entirely  immune. 

When  pyogenic  bacteria  are  confined  to  a  certain  locality  the  process 

is  known  as  a  local  infection.     It  may  be  so  severe  that  a  sufficient 

quantity  of  toxins  are  absorbed  from  the  infected  focus  to  produce 

grave  general  symptoms,  including  more  or  less  high  fever.     There 

13 


194  WOUND  INFECTION  AND  PYOGENIC  BACTERIA 

may  be  even  more  serious  results  when  the  bacteria  enter  the  general 
circulation  through  the  blood  or  lymph  current.  In  this  case  their 
multiplication  and  the  toxin  production  in  the  blood  itself  causes 
grave  symptoms,  generally  including  chills  and  severe  fever  of  an 
irregular  type.  This  process  of  pyogenic  microbes  multiplying  in  the 
general  circulation  is  popularly  known  as  "blood  poisoning,"  and 
technically  as  a  septicemia. 

Another  pathologic  process  which  may  follow  wound  infection 
consists  in  the  formation  of  small  masses  of  bacteria,  perhaps  in  com- 
bination with  small  flocculi  of  pus  or  bits  of  necrotic  tissue.  These 
are  taken  up  by  the  circulation  and  carried  along  in  the  blood  stream 
until  they  finally  become  lodged  in  parts  at  a  considerable  distance 
from  the  original  focus  of  infection.  In  this  manner  the  detached 
masses  of  bacteria  may  be  transported  to  the  lungs,  heart,  liver, 
kidneys,  spleen,  brain,  or  almost  any  one  of  the  internal  organs  of 
the  body  from  foci  of  infection  on  an  extremity  or  some  other  place  on 
the  surface  of  the  body.  After  they  have  finally  become  lodged,  the 
bacteria  multiply  and  may  give  rise  to  secondary  and  generally  mul- 
tiple foci  of  inflammation  and  suppuration.  This  pathologic  occur- 
rence or  process  is  known  as  the  formation  of  multiple  metastatic 
bacterial  emboli  and  the  general  condition  of  blood  poisoning  which 
has  now  become  established  as  a  pyemia  or  septicopyemia.  It  is 
more  dangerous  than  a  septicemia,  and  recovery  is  relatively  rare. 

When  pus  from  an  acute  suppurative  process  is  examined  the 
causative  bacteria  are  generally  found  in  large  numbers.  This 
makes  it  comparatively  easy  to  draw  an  accurate  conclusion  as  to  the 
nature  of  the  particular  infection.  When  more  than  one  species  of 
bacteria  is  present,  either  from  the  beginning  or  very  soon  after, 
the  process  is  known  as  a  mixed  infection.  An  acute  suppurative 
process  due  to  pyogenic  bacteria  may  develop  into  a  subacute  or 
chronic  one.  While  the  inflammation  and  suppuration  may  continue 
the  infecting  bacteria  may  die  out  and  disappear.  The  pus  then 
becomes  void  of  living  bacteria  and  is  known  as  sterile  pus.  More  or 
less  extensive  masses  of  necrotic  tissue  which  are  always  a  source  of 
inflammatory  irritation  may,  however,  cause  the  inflammation  and 
suppuration  to  continue.  When  necrotic  tissue  is  present,  it  may 
become  the  soil  for  the  development  of  purely  saprophytic  bacteria. 
Gangrene  may  set  in,  and  if  a  sufficient  quantity  of  the  putrefactive 
products  is  absorbed  a  condition  of  sapremia  may  supervene. 


STAPHYLOCOCCUS  PYOGENES. 

The  most  common  pus-producing  bacteria  in  man  and  animals 
are  the  pyogenic  staphylococci. 

Varieties. — According  to  the  pigments  formed  by  these  bacteria 
they  are  distinguished  as: 


MORPHOLOGY  AND  STAINING  PROPERTIES 


195 


Staphylococcus  pyogenes  aureus  (golden  yellow  pigment). 
Staphylococcus  pyogenes  albus  (white  pigment). 
Staphylococcus  pyogenes  citreus  (lemon  yellow  pigment). 
They  are  given  in  the  order  of  their  virulency,  the  aureus  being  the 
most,  the  citreus  the  least  virulent. 


FIG.  110 


FIG.  Ill 


Staphylococcus  pyogenes  aureus.     X  1000. 
(Author's  preparation.) 


Staphylococcus  pyogenes  albus.     X  1000. 
(Author's  preparation.) 


Occurrence  and  Pathogenesis. — Pyogenic  staphylococci  are  found 
everywhere  in  the  outside  world,  but  are  most  numerous  on  the  skin 
of  man  and  animals.  They  are  also  frequently  found  in  the  feces. 
They  vary  much  in  virulency,  and  are  most  virulent  when  they  come 
directly  from  a  badly  infected  wound  or  a  case  of  septicemia  or 
pyemia.  Surgeons  operating  on  pus  cases  may  easily  and  frequently 
do  fatally  infect  themselves.  The  staphylococci,  particularly  the 
Staphylococcus  pyogenes  aureus,  are  the  cause  of  all  varieties  of 
wound  infections  such  as  septicemia,  pyemia,  endocarditis,  septic 
pneumonia,  puerperal  fever,  bone  diseases,  etc. 

Morphology  and  Staining  Properties. — In  the  hanging  drop  the 
Staphylococcus  pyogenes  shows  a  completely  globular  or  spherical 
shape.  It  exhibits  a  lively  Brownian  molecular  motion.  It  stains 
well  with  the  ordinary  watery  basic  anilin  dies.  When  not  stained 
too  deeply  it  frequently  shows  a  line  in  the  middle  dividing  it  into 
two  equal  hemispheres.  This  line  indicates  the  site  where  division 
will  occur  when  the  organism  multiplies.  The  organism  is  Gram 
positive.  The  individual  cocci  vary  from  0.7  to  0.9  of  a  micron  in 
diameter,  and  sometimes  have  a  diameter  up  to  1.2  micron.  The 
Staphylococcus  pyogenes  albus  and  citreus  are  frequently  larger  than 
the  aureus,  but  the  size  depends  largely  upon  the  culture  medium. 
The  cocci  are  generally  smaller  in  young,  rapidly  growing  cultures, 
and  larger  in  old,  slowly  growing  ones.  In  cover-glass  preparations 


196  WOUND  INFECTION  AND  PYOGENIC  BACTERIA 

from  artificial  cultures  the  organisms  occur  in  large,  irregular  grape- 
like  masses.  In  pus,  however,  this  seldom  occurs;  instead  they  are 
generally  found  in  small  groups  or  pairs,  double  pairs  (tetrads),  or 
sometimes  short  chains.  This  sometimes  makes  it  impossible  to  decide 
quickly  whether  the  organism  is  a  staphylococcus,  a  diplococcus,  or 
a  streptococcus.  Cultures,  of  course,  will  definitely  identify  the 
organism.  The  staphylococci  possess  no  flagella,  and  do  not  form 
spores. 

Cultural  and  Biological  Properties. — Pyogenic  staphylococci  grow 
on  a  great  variety  of  artificial  culture  media.  Their  range  of  tem- 
peratures is  wide  and  lies  between  9°  and  42°  C.;  the  optimum  tem- 
perature is  at  24°  to  28°  C.,  not  at  blood  temperature.  They  grow 
both  in  the  presence  and  absence  of  oxygen,  and  also  in  a  hydrogen 
atmosphere,  but  not  in  pure  carbon  dioxide  or  illuminating  gas.  The 
reaction  of  the  culture  soil,  like  the  temperature,  may  vary  consider- 
ably, but  a  slight  alkalinity  is  most  favorable  to  their  growth.  The 
organisms  multiply  very  rapidly  in  nutrient  bouillon,  and  under 
favorable  conditions  a  tube  inoculated  in  the  ordinary  manner  may, 
after  twenty-four  hours,  contain  85,000,000  cocci  per  cubic  centi- 
meter. The  bouillon  soon  becomes  intensely  clouded,  sometimes  a 
slight  pellicle  develops  on  the  surface,  and  a  slimy  sediment  is  always 
formed  at  the  bottom  of  the  tube  after  a  growth  of  several  days. 
The  tubes  have  a  strong  smell  of  old  starch  paste.  The  organism 
also  grows  well  in  Dunham's  peptone  water  and  in  milk.  The  latter 
shows  coagulation  sometimes  after  a  few  days,  always  after  eight 
days.  In  gelatin  stab  cultures  the  growth  extends  along  the  entire 
stab,  and  after  a  few  days  liquefaction  begins  at  the  surface  and  pro- 
gresses downward.  On  gelatin  plates  small  yellow  points  -around 
which  the  medium  is  liquefied  appear  after  two  days.  On  blood  serum 
and  LoefHer's  blood-serum  mixture  the  growth  liquefies  the  medium 
very  slowly,  sometimes  not  at  all.  If  fresh  sterile  blood,  particularly 
rabbit's  blood,  is  added  to  a  solid  culture  medium  on  which  the 
staphylococcus  is  subsequently  grown,  solution  of  the  red  blood  cor- 
puscles (i.e.,  hemolysis)  occurs.  Potatoes  are  a  good  culture  medium 
for  the  organism,  which  does  not  ferment  sugar. 

Pigment  formation  requires  the  presence  of  oxygen.  It  is  not  well 
marked  in  the  presence  of  too  much  strong  diffuse  light,  but  shows 
best  when  the  cultures  receive  only  little  light  and  are  kept  at  tem- 
peratures between  20°  to  22°  C. 

The  liquefying  properties  of  the  staphylococci  depend  upon  the 
secretion  of  a  tryptic  ferment.  It  is  now  claimed  that  the  strong 
ferment  liquefying  gelatin  and  the  rather  weak  one  liquefying  blood 
serum  and  egg-albumen  are  two  distinct  types.  Hemolysis  due  to 
the  growth  of  the  organism  is  caused  by  a  hemolysin.  Staphy- 
lococci also  produce  a  substance  which  is  very  injurious  to  the  leu- 
kocytes of  the  rabbit.  This  body  has  been  called  leukocidin,  which 
means  killing  white  blood  corpuscles.  Several  investigators  claim 


VACCINE  THERAPY  197 

to  have  been  able  to  produce  amyloid  substance1  in  the  internal  organs 
of  animals  treated  with  prolonged  systemic  infections  of  pure  cultures 
of  staphylococci. 

Experiments  with  Staphylococci. — Many  investigators  have  experi- 
mented upon  themselves  with  staphylococci  and  animal  experiments 
without  number  have  been  made.  The  results^show  that  man  is  more 
susceptible  to  the  detrimental  effects  than  any  of  the  lower  animals. 
The  difference  in  virulency  in  the  Staphylococcus  pyogenes  aureus, 
albus,  and  citreus  can  be  very  well  exhibited  by  injecting  small  amounts 
of  bouillon  cultures  into  the  anterior  chamber  of  the  eye  of  a  number 
of  rabbits.  The  aureus  generally  causes  a  very  violent  suppurative 
panophthalmitis  (inflammation  of  the  entire  eyeball),  destroying  the 
eye,  and  sometimes  leads  to  a  general  infection  (septicopyemia)  and 
death.  The  albus  produces  a  less  serious  inflammation  and  the 
citreus  a  very  mild  one. 

Resistance  of  the  Organisms. — The  resistance  of  different  strains  of 
staphylococci  toward  inimical,  physical,  and  chemical  agents  varies 
considerably.  Sternberg  killed  cultures  by  an  exposure  to  a  temper- 
ature of  62°  C.  for  ten  minutes  and  80°  C.  for  1 J  minutes.  Others 
have  had  cultures  which  could  withstand  60°  C.  for  one  hour  without 
being  killed.  The  resistance  of  these  cocci  evidently  is  increased  by 
previous  drying  out,  particularly  in  pus.  An  exposure  for  thirty 
to  sixty  minutes  at  80°  C.,  however,  apparently  kills  the  pathogenic 
staphylococci  under  all  conditions.  Repeated  freezing  alternating 
with  thawing  seems  to  have  no  effect  whatever  upon  the  organism. 
Direct  sunlight  does  not  appear  to  kill  the  staphylococci  even  after 
several  hours.  Drying  has  little  effect,  and  kills  them  only  after  many 
weeks.  The  effect  of  antiseptics  and  germicides  upon  pyogenic  bac- 
teria is  as  follows :  Solution  of  corrosive  sublimate  1  to  1000  kills  the 
staphylococci  in  thirty  to  sixty  minutes;  chloroform  vapors  in  twenty 
minutes;  iodoform  has  no  effect  whatever;  absolute  alcohol  has  no 
effect  upon  dried  cocci,  but  50  per  cent,  alcohol  kills  them  in  ten 
minutes;  3  per  cent,  carbolic  acid  in  two  to  two  and  one-half  minutes; 
1  per  cent,  solution  of  formalin  in  twenty-four  hours;  even  a  5  per 
cent,  solution  of  formalin  only  kills  after  thirty  to  thirty-five  minutes. 
Some  of  the  anilin  stains,  like  methyl  violet,  even  in  very  weak  solu- 
tions, kill  staphylococci  very  quickly. 

Vaccine  Therapy. — Chronic  staphylococcus  infections  of  a  low  type, 
such  as  furuncles,  discharging  sinuses,  old  abscesses,  etc.,  have  been 
found  capable  of  improvement,  and  often  of  cure,  by  vaccine  or  bac- 
terine  treatment.  An  autogenous  vaccine,  i.  e.,  one  from  a  pure  culture 
obtained  from  the  infected  patient,  gives  the  best  results  in  these 

1  Amyloid  material  or  substance  is  one  of  the  hyaline  materials,  which  are  degenerative, 
pathologic  products.  It  has  certain  characteristic  staining  properties  and  certain  color  re- 
actions like  starch  (amylum).  It  is,  however,  not  a  carbohydrate  like  starch,  but  a  proteid 
body.  It  is  first  formed  or  deposited  in  the  walls  of  the  small  vessels  of  the  internal  organs, 
particularly  the  spleen,  liver,  and  kidneys. 


198 


WOUND  INFECTION  AND  PYOGENIC  BACTERIA 


cases.  The  killed  staphylococci  are  injected  in  proper  doses  at 
intervals  of  about  six  to  eight  days.  If  it  is  too  troublesome  to  prepare 
an  autogenous  vaccine,  a  stock  vaccine,  as  prepared  by  pharma- 
ceutical houses,  may  be  used. 


STREPTOCOCCUS  PYOGENES. 

Occurrence  and  Pathogenesis. — Pyogenic  streptococci  are  evidently 
not  found  as  commonly  in  the  outside  world  as  staphylococci,  nor  do 
they  appear  to  thrive  as  well  as  saprophytes.  The  Streptococcus 
pyogenes  is  the  cause  of  suppurative  processes  of  all  kinds,  such  as 
septicemia,  pyemia,  puerperal  infection,  erysipelas,  etc.  General 
infections  by  the  Streptococcus  pyogenes  are,  as  a  rule,  even  more 
virulent  than  those  of  the  staphylococcus. 


FIG.  112 


FIG.  113 


Streptococci  from  a  pure  culture  in  bouillon.       Streptococci  from   human  pus,  Gram's  stain. 
X  1000.     (Kolle  and  Wassermann.)  X  1000.     (Author's  preparation.) 

Morphology  and  Staining  Properties. — The  Streptococcus  pyogenes 
is  non-motile,  has  no  flagella,  does  not  form  spores,  and  can  grow  in 
the  presence  or  absence  of  oxygen.  The  individual  cocci  vary  from 
0.4  to  1  micron  in  diameter.  The  length  of  the  chains  differs  so 
much  that  varieties  such  as  the  Streptococcus  pyogenes  longus  and  the 
Streptococcus  pyogenes  brevis,  the  long  and  the  short  chain  cocci, 
have  been  distinguished.  The  individual  cocci  forming  the  chain 
vary  not  only  in  size  but  in  shape.  Some  are  entirely  spherical;  others 
are  flattened  at  both  poles,  so  that  the  chain  seems  to  consist  of 
disk-like  bodies;  still  others  are  flattened  out  laterally,  making  the 
individual  cocci  oval  or  like  bacilli  with  pointed  ends.  When  the  cocci 
grow  in  the  long  axis  of  the  chain  with  infrequent  division,  chains  are 
formed  which  look  much  like  streptobacilli,  i.  e.,  bacilli  in  the  form  of 
chains.  The  formation  of  short  or  long  chains  is  not  an  absolutely 


CULTURAL  AND  BIOLOGIC  PROPERTIES  199 

permanent  characteristic  of  certain  varieties.  It  often  depends  upon 
differences  in  occurrence,  environment,  and  culture  media.  Certain 
stems  of  streptococci,  however,  generally  show  a  tendency  to  form  short 
chains  of  only  four,  six,  or  eight  cocci,  while  others  have  the  opposite 
tendency.  Virulent  forms  in  tissues  often  appear  in  short  chains; 
these  generally  have  a  capsule.  The  Streptococcus  pyogenes  stains 
well  with  the  ordinary  watery  anilin  dyes,  and  keeps  Gram's  stain. 
There  are,  however,  other  pathogenic  streptococci,  such  as  the 
streptococcus  found  in  abscess  of  the  udder  in  cows  (Nocard's 
streptococcus),  that  are  Gram  negative,  as  are  also  some  saprophytic 
streptococci. 

Cultural  and  Biologic  Properties. — Pyogenic  streptococci  grow  and 
exhibit  the  typical  chain  form  much  better  in  fluid  than  in  solid  cul- 
ture media.  The  Streptococcus  pyogenes  longus  clouds  the  bouillon 
diffusely;  the  Streptococcus  pyogenes  brevis  produces  less  clouding. 
A  faintly  alkaline  reaction  of  the  nutrient  bouillon  is  best.  An  in- 
crease of  the  peptone  of  the  bouillon  from  1  to  3  to  5  per  cent,  and 
the  addition  of  0.2  to  1  per  cent,  glucose  is  also  favorable  to  the  growth 
of  the  organism.  Too  much  glucose,  however,  lessens  the  virulency 
of  the  streptococcus  and  causes  it  to  die  out  sooner,  since  the  acid 
formed  from  the  sugar  changes  the  reaction  of  the  culture  soil. 

Blood  serum,  generally  used  with  the  addition  of  one  part  of  nutri- 
ent bouillon  to  three  parts  of  serum,  is  an  excellent  medium  for  the 
growth  of  pyogenic  streptococci  with  preservation  of  their  virulency. 
Human,  rabbit,  or  horse  serum  may  be  used.  The  serum  must  not 
be  solidified,  as  the  organism  does  not  grow  very  abundantly  on  solid 
media.  On  agar  plates  kept  in  the  incubator,  small  grayish  or  yel- 
lowish gray,  finely  granular  colonies  develop,  which  generally  do  not 
exceed  0.5  mm.  in  size.  The  deep  colonies  are  brownish,  round,  or 
oval.  Agar  streak  and  stick  cultures  are  not  characteristic.  Gelatin 
kept  at  20°  to  22°  C.  is  generally  not  liquefied,  but  gelatin  which  can 
be  kept  at  29°  C.  is  sometimes.  Saprophytic  streptococci,  cultivated 
from  dust  and  from  the  contents  of  the  intestines,  liquefy  gelatin. 
Pyogenic  streptococci  only  occasionally  grow  on  potatoes.  The 
optimum  temperature  of  growth  is  in  the  neighborhood  of  the  blood 
temperature.  At  12°  to  15°  C.  the  development  is  poor,  at  24°  C.  good, 
and  best  at  35°  to  37°  C. ;  at  40.5°  C.  it  falls  off  and  at  42.3°  C.  it  ceases 
entirely.  Slightly  different  figures  are  given  by  other  authors.  Strep- 
tococci can  grow  in  the  presence  of  oxygen,  but  they  do  not  require  it, 
and  in  fact  some  varieties  evidently  grow  better  anaerobically.  All 
streptococci  form  acid  in  their  growth,  chiefly  lactic  acid,  and  coagu- 
late milk,  in  which  they  generally  grow  poorly  because  of  the  acid 
formation.  Some  stems  form  a  brownish-yellow  pigment,  partic- 
ularly in  gelatin  and  in  the  sediment  of  bouillon  cultures.  While 
pyogenic  streptococci  generally  grow  for  two  to  three  days  only  on 
artificial  culture  media  and  then  die  out,  certain  stems  may  remain 
alive  longer,  occasionally  for  several  weeks. 


200  WOUND  INFECTION  AND  PYOGENIC  BACTERIA 

Resistance. — The  Streptococcus  pyogenes  generally  dies  quickly 
in  liquid  cultures  or  when  dried  out  on  silk  threads.  When  con- 
tained in  dry  pus  it  sometimes  remains  alive  for  weeks  and  months, 
particularly  if  the  cocci  have  dried  out  slowly  and  are  subsequently 
kept  at  low  temperatures  and  protected  against  light.  They  are 
comparatively  resistant  against  high  temperatures,  generally  being 
able  to  withstand  heat  of  60°  G.  for  one  hour  and  occasionally  for 
two  hours;  70°  to  75°  C.  acting  for  one  hour,  however,  positively  kills 
them.  Cold  appears  to  have  no  effect.  A  number  of  antiseptics 
have  been  tried  by  von  Lingelsheim.  He  gives  the  following  concen- 
trations as  killing  Streptococcus  pyogenes  in  fifteen  minutes: 

Hydrochloric  acid     .      /    »      .      .      .      .    ".      .      ,      .  to  150 

Sulphuric  acid     .      .      .      .      .      . ',.      .      .      .      .      »  to  150 

Ammonia .  ••  ;      .      .      .      .      .  to  15 

Corrosive  sublimate        .      .      .      .      .      .      ,      .      .      .  to  1500 

Copper  sulphate to  125 

Chloride  of  iron    .      .      »      .      .      ...      ....  1  to  350 

Carbolic  acid        .      .      .      *      .      .      .      .      .....  to  200 

Kresol .      .      .    *.      .      .      .      .  to  175 

Lysol ....*,..;.  to  200 

Kreolin .      .      .      .  1  to  80 

Vaccine  Therapy. — This  appears  to  be  valueless  in  the  acute  infec- 
tions, but  in  slow  chronic  cases  the  bacterine  treatment  often  leads  to 
good  results. 

BACILLUS  PYOCYANEUS. 

Occurrence  and  Pathogenesis. — The  Bacillus  pyocyaneus  was  first 
found  in  and  isolated  from  green  pus.  It  is,  however,  also  very 
common  as  a  saprophyte  in  the  outside  world.  It  has  been  found  in 
sewage,  manure,  water,  the  intestinal  contents  of  man  and  domestic 
animals  (particularly  the  hog),  rooms  and  hospital  wards,  barns, 
etc.  It  generally  lives  as  a  harmless  commensale  in  the  intestines 
of  man  and  animals,  but  it  has  been  known  in  ill-nourished,  weak 
children  to  invade  the  organism  from  the  intestine  and  in  a  few  cases 
to  have  led  to  a  general  septicemia  and  death.  It  is  frequently  the 
cause  of  local  suppurations,  also  of  purulent  middle-ear  inflamma- 
tions. It  imparts  a  green  color  to  the  pus.  The  Bacillus  pyocyaneus  is 
also  found  in  pus  in  animals,  but  it  is  doubtful  whether  it  alone  can  start 
a  suppurative  inflammation  in  domestic  animals.  According  to  Baru- 
chelli,  injection  of  the  Bacillus  pyocyaneus  into  the  peritoneal  cavity 
of  a  male  guinea-pig  occasionally  produces  a  periorchitis  which  may 
cause  it  to  be  confounded  with  the  Bacillus  mallei  in  Strauss'  biologic 
test  for  glanders  (see  Chapter  XXVI  on  the  Glanders  Bacillus). 

Morphology  and  Staining  Properties. — The  Bacillus  pyocyaneus  is 
generally  a  small,  slender  bacillus,  but  it  also  occurs  in  larger,  plumper 
varieties  and  the  measurements  given  by  various  authors  are  from 
0.3  to  1  to  0.6  to  2  to  6  micra.  It  has  rounded  ends  and  often  forms 
short  chains  of  a  few  individual  bacilli;  longer  pseudofilaments  are 
rare.  The  bacillus  is  very  actively  motile  and  possesses  a  flagellum 


TOXINS 


201 


FIG.  114 


at  one  end.  After  long-continued  culture  on  artificial  media  the 
flagellum  may  be  lost,  but  it  reappears  after  inoculation  into  an 
animal.  It  does  not  form  spores.  It  stains  well  with  the  watery 
anilin  dyes,  but  is  Gram  negative. 

Cultural  and  Biologic  Properties. — The  Bacillus  pyocyaneus  grows 
well  at  room  and  incubator  temperature.  On  gelatin  plates  small 
yellowish-white  colonies  appear  first  in  the  deeper  parts  and  extend 
rapidly  toward  the  surface,  where  they  then  show  a  dark  yellow  centre 
and  a  periphery  with  radial  striation.  The  medium  itself  assumes 
a  typical  greenish  fluorescent  tint  around  the  colonies.  The  gelatin 
is  liquefied  and  the  culture  sinks  to  the  bottom,  forming  a  slimy  red- 
brownish  mass.  In  gelatin  stick 
cultures  liquefaction  first  appears 
at  the  surface  in  a  funnel-shaped 
manner,  and  then  rapidly  spreads 
downward.  On  agar  slants  kept 
at  incubator  temperature  the 
growth  is  rapid,  and  the  green 
pigment  generally  turns  brownish 
after  two  days,t  spreading  succes- 
sively through  the  entire  culture 
medium.  Pigment  formation  oc- 
curs only  in  the  presence  of 
oxygen,  and  the  liveliest  colors 
are  formed  at  room,  but  not  at 
incubator,  temperatures.  The 
bacillus  not  infrequently  produces 
a  blue  or  even  from  the  begin- 
ning a  greenish-brown  pigment 
instead  of  a  decided  green  color. 

In  bouillon  the  bacillus  grows  well.  A  white  ring  first  shows  at  the 
margin  of  the  free  surface,  and  from  it  a  complete  pellicle  is  formed. 
From  the  latter  a  green  zone  extends  downward  into  the  medium. 
After  two  weeks  the  whole  growth  sinks  to  the  bottom  and  forms 
a  slimy  sediment.  After  many  weeks  in  the  incubator  the  formation 
of  an  autolytic  ferment  causes  the  growth  to  undergo  self -digestion. 
Milk  is  coagulated  by  the  bacillus.  The  organism  grows  well  on 
potatoes,  and  forms  first  a  grayish-brown  and  later  on  a  yellowish- 
green  pigment,  which  is  composed  of  two  constituent  bodies.  One  of 
these,  known  as  pyocyanin,  is  bluish  green  and  soluble  in  chloroform, 
the  other  is  greenish,  fluorescent,  and  insoluble  in  chloroform  or 
alcohol,  but  soluble  in  water.  Pyocyanin  is  originally  a  colorless  sub- 
stance, and  obtains  its  color  only  after  subsequent  oxidation. 

Toxins. — The  Bacillus  pyocyaneus  is  pathogenic  in  experimental 
inoculation  for  guinea-pigs  and  goats;  rabbits,  mice,  and  pigeons  are 
slightly  susceptible.  Wassermann  has  shown  that  the  pathogenic  effect 
of  the  organism  upon  man  and  animals  depends  upon  a  soluble  toxin 
and  an  insoluble  endotoxin. 


Bacillus  pyocyaneus.      X  1000.     (Author's 
preparation.) 


202  WOUND  INFECTION  AND  PYOGENIC  BACTERIA 

Resistance. — The  Bacillus  pyocyaneus  is  quite  resistant.  It  cannot 
be  readily  killed  by  drying  out,  and  its  behavior  toward  antiseptics 
and  germicides  is  similar  to  that  of  the  pyogenic  staphylococci. 

QUESTIONS. 

1.  Explain  the  term  pyogenic  bacteria. 

2.  Which  are  the  most  common  pyogenic  microorganisms? 

3.  Where  are  they  found? 

4.  What  does  the  term  ubiquitous  mean? 

5.  Are  pyogenic  bacteria  most  commonly  found  as  parasites? 

6.  What  is  meant  by  wound  healing  by  primary  intention? 

7.  What  is  meant  by  a  local;  what  by  a  general  pyogenic  infection? 

8.  What  is  a  septicemia? 

9.  What  are  multiple  metastatic  bacterial  emboli? 

10.  What  is  a  septicopyemia? 

11.  What  is  a  mixed  infection? 

12.  What  is  a  sapremia? 

13.  Name- the  three  different  varieties  of  the  common  pus-forming  cocci? 

14.  Name  a  number  of  diseases  caused  by  the  pyogenic  staphylococci. 

15.  Describe  the  Staphylococcus  pyogenes  aureus  in  a  hanging  drop  and  in  a 
stained  cover-glass  preparation. 

16.  How  does  this  coccus  act  when  stained  by  Gram's  method?    Describe 
this  staining  method. 

17.  How  does  the  Staphylococcus  pyogenes  generally  present  itself  in  pus? 

18.  At  what  temperature  does  this  coccus  grow?   What  is  its  optimum  temper- 
ature?   What  is  its  action  toward  free  oxygen? 

19.  Describe  a  bouillon  culture  and  a  gelatin  stab  culture    of    the  Staphy- 
lococcus pyogenes  aureus. 

20.  What  is  meant  by  the  hemolytic  property  of  the  Staphylococcus  pyogenes? 

21.  What  is  leucocidin? 

22.  What  is  amyloid  material  or  substance? 

23.  Discuss  the  resistance  of  the  Staphylococcus   pyogenes  toward  physical 
and  chemical  agencies. 

24.  What  is  the  effect  of  vaccine  therapy  in  chronic  Staphylococcus  infection? 
Describe  the  principle  and  details  of  Staphylococcus  vaccine  therapy. 

25.  Give  the  morphologic  features  of  the  Streptococcus  pyogenes. 

26.  What  is  the  difference  between  Streptococcus  pyogenes  longus  and  Strepto- 
coccus pyogenes  brevis? 

27.  What  are  the  staining  properties  of  the  Streptococcus  pyogenes? 

28.  Name  some  Gram-negative  streptococci. 

-29.  What  kind  of  media  are  best  adapted  to  the  growth  of  Streptococcus 
pyogenes? 

30.  What  effect  have  peptone  and  sugar  upon  the  growth  of  the  Streptococcus 
pyogenes? 

31.  What  culture  medium  is  best  for  preserving  the  virulency  of  Streptococcus 
pyogenes? 

32.  Describe  the  colonies  of  the  streptococcus  on  agar  plates. 

33.  Under  what  conditions  does  the  streptococcus  liquefy  gelatin? 

34.  What  is  its  optimum  temperature?    What  is  its  relation  to  free  oxygen? 

35.  Discuss  the  resistance  of  the  streptococcus. 

36.  Describe  the  occurrence  and  pathogenesis  of  the   Bacillus  pyocyaneus. 
Why  is  it  called  pyocyaneus? 

37.  Describe  the  morphologic  features  of  this  bacillus. 

38.  Does  it  belong  to  the  amphitrichse  or  peritrichae? 

39.  What  effect  does  an  intraperitoneal  injection  of  the  bacillus  into  a  male 
guinea-pig  sometimes  have?    Why  is  it  important  to  remember  this? 

40.  Describe  a  gelatin  plate  and  a  gelatin  stick  culture  of  the  organism. 

41.  Describe  a  bouillon  culture. 

42.  What  is  meant  by  auto  lysis  and  an  autolytic  ferment  secreted  by  the 
Bacillus  pyocyaneus? 

43.  What  are  the  properties  of  the  two  pigments  formed  by  the  Bacillus 
pyocyaneus? 

44.  What  kind  of  toxins  does  this  bacillus  form? 

45.  Discuss  the  resistance  of  the  organism. 


CHAPTER    XVII. 

PYOGENIC    BACTERIA    IN    DOMESTIC   ANIMALS— STREPTOCOCCUS 

EQUI_STREPTOCOCCI  IN  MORBUS,  MACULOSUS  EQUI,  AND 

PLEUROPNEUMONIA    IN   HORSES— BOTRYOCOCCUS 

ASCOFORMANS— PYOGENIC  BACTERIA  IN 

CATTLE— BACILLUS  PYOGENES  SUIS. 

THE  common  pyogenic  bacteria  described  in  the  preceding  chapter 
are  in  man,  in  the  majority  of  cases,  the  cause  of  suppurative  inflam- 
mations, septicemia  and  pyemia.  They  are  also  very  frequently 
responsible  for  the  identical  pathological  processes  in  domestic  animals. 
Karlinski  investigated  a  large  number  of  pyogenic  affections  in  man 
and  animals  and  found  the  various  causative  bacteria  as  follows : 


In  man 

Streptococci 

Cases. 
45 

Staphylococci 

144 

In  man 
In  mammals 

Other  bacteria      . 
Streptococci 

15 
23 

In  mammals 

Staphylococci 

45 

In  mammals 

Other  bacteria 

15 

In  birds 

Streptococci 

11 

In  birds 

Staphylococci        . 

40 

In  birds 

Other  bacteria 

20 

Lucet  made  a  bacteriological  examination  of  93  cases  of  suppura- 
tions of  various  types  in  horses  and  found  Staphylococci  in  86  cases. 
The  three  varieties  were  found  either  in  pure  or  mixed  cultures,  but 
the  Staphylococcus  pyogenes  albus  appeared  more  commonly  as  the 
cause  of  suppuration  in  the  horse  than  the  Staphylococcus  pyogenes 
aureus.  In  man  the  contrary  is  true.  Schiitz,  Jensen,  and  Nocard 
similarly  found  Staphylococci  in  most  cases  of  suppuration  in  the 
horse,  and  Streptococcus  pyogenes  also  occurred  in  a  number  of  cases. 
Pyogenic  Staphylococci  and  streptococci  have  also  been  found  in 
pyogenic,  metastatic  joint  affections  in  horses,  particularly  when 
young;  occasionally  as  the  cause  of  suppuration  and  erysipelas  in 
cattle,  and  also  in  puerperal  fever  in  cows.  Ordinarily,  cattle  do 
not  seem  very  susceptible  to  infection  by  these  pyogenic  bacteria. 
Dogs,  however,  are  more  susceptible.  Streptococcus  pyogenes  has 
further  been  found  as  the  cause  of  obstinate  eczema  of  the  tail  of 
the  horse. 

A  number  of  bacteria  acting  as  specific  pyogenic  microbes  for 
certain  domestic  animals  will  now  be  considered  briefly. 


204  PYOGENIC  BACTERIA  IN  DOMESTIC  ANIMALS 


STREPTOCOCCUS  EQUI. 

Occurrence  and  Pathogenesis. — The  Streptococcus  equi  was  first  seen 
by  Rivolta  in  1873,  and  identified  as  the  cause  of  strangles  in  the 
horse  by  Schiitz  in  1888,  and  also  by  Sand  and  Jensen,  independently, 
at  about  the  same  period.  The  specific  disease  which  it  causes  is 
also  known  as  Coryza  contagiosa  equorum,  distemper;  "Druse  der 
Pferde,"  in  German;  and  "Gourme,"  in  French.  Strangles  is  a  febrile, 
infectious,  mucopurulent  affection  of  the  nasal  and  buccal  mucosa, 
with  abscess  formation  in  the  laryngeal  and  retropharyngeal  lymph 
glands.  The  Streptococcus  equi  is  found  in  the  nasal  discharge  and 
in  the  abscesses  in  the  affected  lymph  glands;  in  the  latter  often  in 
pure  culture,  in  the  former,  of  course,  mixed  with  other  bacteria. 
It  sometimes  causes  a  simple  nasal  catarrh  without  suppurative 
processes  in  the  glands,  and  at  other  times  suppurative  pleuritis  and 
exanthematous  affections,  with  vesicle  and  pustule  formation.  It  may 
also  cause  septicemia  and  pyemia.  Metastases  have  been  found  in 
the  brain,  liver,  spleen,  kidneys,  mesenteric  gland's,  and  intestines,  etc. 

Morphology  and  Staining  Properties. — The  Streptococcus  equi  is 
composed  of  cocci  which  may  be  spherical  or  oval  or  perfectly  cylin- 
drical short  disks.  The  latter  arrangement  is  often  seen  in  tissues, 
and  the  disks  may  be  so  crowded  that  the  dividing  lines  of  the  indi- 
vidual cocci  are  invisible,  causing  the  organism  to  appear  like  a  curved 
filament  rather  than  a  true  streptococcus.  The  chains  are  usually 
very  long,  and  may  contain  from  fifty  to  one  hundred  cocci;  they  are 
rarely  straight,  but,  as  a  rule,  curved  and  twisted.  Short  chains  also 
occur,  and  sometimes  even  diplococci  and  single  cocci.  The  organism 
stains  with  the  ordinary  watery  anilin  stains,  and  is  Gram  positive. 
The  washing  in  alcohol,  however,  must  not  be  continued  for  too 
long  a  period,  as  it  may  cause  decolorization  of  the  organism.  Some 
observers  claim  that  the  Streptococcus  equi  does  not  stain  by  Gram's 
method. 

Cultural  and  Biologic  Properties. — The  Streptococcus  equi  grows  in 
the  presence  or  absence  of  free  oxygen,  and  at  room  or  incubator 
temperature.  In  bouillon  the  growth  forms  fine  flocculi,  which  fall  to 
the  bottom  of  the  tube  and  later  develop  a  sediment,  leaving  the 
upper  strata  of  the  medium  clear.  In  gelatin  stick  cultures  very  small 
punctiform  white  colonies  are  formed  along  the  stab.  On  agar  slants 
or  plates  the  colonies  reach  the  size  of  a  pinhead.  They  are  grayish, 
not  transparent,  do  not  become  confluent,  and  adhere  firmly  to  the 
medium.  On  solidified  blood  serum  the  colonies  are  glassy  and 
translucent,  and  become  confluent  at  a  later  stage.  In  the  condensed 
water  of  blood-serum  tubes  a  fine  precipitate  composed  of  very  long 
chains  is  found.  Some  authors  claim  that  there  is  no  growth  on 
potatoes,  others  state  that  a  grayish,  slimy  growth  is  present  after 
eight  days.  The  organism  does  not  ferment  sugar;  on  artificial  media 


OCCURRENCE  AND  PATHOGENESIS  205 

it  sometimes  forms  pseudofilaments.     It  is  pathogenic  to  mice,  and 
according  to  Rabe,  also  to  guinea-pigs. 

STREPTOCOCCI  IN  OTHER  DISEASES. 

Streptococci  in  Morbus  Maculosus  Equorum. — The  disease  known  as 
morbus  maculosus  equorum,  acute  hemorrhagic  anasarcous  toxemia, 
or  petechial  fever,  occurs  either  sporadically  or  in  epidemic  form, 
particularly  after  influenza  and  strangles.  Ligniere,  who  investigated 
the  bacteriology,  claims  that  he  generally  found  the  Streptococcus 
pyogenes  and  more  rarely  the  Streptococcus  equi  and  the  Bacillus 
equisepticus  in  the  blood  of  horses  which  had  died  from  the  disease. 
However,  neither  Ligniere  nor  any  other  investigator  has  been  able 
to  produce  the  disease  experimentally  by  the  inoculation  of  these 
organisms. 

Streptococci  in  Contagious  Pleuropneumonia  of  Horses. — An  organism 
which  presents  itself  as  a  diplococcus  in  tissues,  but  as  a  streptococcus 
in  pure  cultures,  was  described  as  the  cause  of  this  disease  by  Schiitz 
in  1887.  It  often  shows  a  capsule  in  tissues,  stains  with  the  ordinary 
watery  anilin  stains,  but  not  by  Gram's  method.  It  grows  on  agar 
and  gelatin  at  room  and  incubator  temperature,  and  Schiitz  claims 
that  he  has  been  able  to  produce  the  disease  in  horses  by  intrathoracic 
injections  of  pure  culture.  According  to  Ligniere,  Schutz's  organism 
is  identical  with  the  Streptococcus  equi. 

Streptococci  in  Apoplectiform  Septicemia  in  Chickens. — Noergaard 
and  Mohler  have  described  a  streptococcus  occurring  in  short  or 
long  chains,  with  individual  cocci  0.6  to  0.8  micron  in  diameter,  as 
the  etiologic  factor  of  this  disease.  The  organism  is  Gram  positive. 
It  grows  in  the  presence  or  absence  of  oxygen.  It  forms  flaky  masses 
in  bouillon,  and  leaves  the  fluid  clear,  changes  an  alkaline  medium  to 
an  acid  one,  and  does  not  liquefy  gelatin.  It  does  not  greatly  change 
the  appearance  of  milk,  nor  does  it  form  a  visible  growth  on  potatoes. 
It  is  fatal  to  fowls,  mice,  rabbits,  and  swine,  but  not  to  guinea-pigs, 
dogs,  and  sheep. 

BOTRYOCOCCUS  ASCOFORMANS. 

Occurrence  and  Pathogenesis. — The  organism,  now  generally  known 
as  Botryococcus  ascoformans,  is  the  cause  of  a  suppurative  infection 
known  as  botryomycosis.  The  clinical  manifestations  and  histo- 
pathology  of  the  disease  somewhat  resemble  actinomycosis,  but  the 
causative  organism  is  entirely  distinct.  While  actinomycosis  is  com- 
mon in  cattle  and  rare  in  the  horse,  botryomycosis  is  common  in  the 
horse  and  relatively  rare  in  cattle.  It  is  also  occasionally  seen  in  the 
hog  and  in  man.  The  disease  is  remarkable  because  it  represents  a 
pathologic  process  showing  some  common  features  of  an  infectious 
granulonia  and  of  a  true  tumor.  The  botryomycotic  new  formations 


206  PYOGENIC  BACTERIA  IN  DOMESTIC  ANIMALS 

represent  grayish-white,  fibrous,  lardaceous  connective-tissue  masses 
in  which  smaller  areas  of  cellular  vascular  granulation  tissue  are  found, 
often  with  small  cavities  and  fistulous  tracts  containing  varying 
amounts  of  pus,  and  in  the  latter  the  peculiar  zoogleal  masses  of  the 
Micrococcus  ascoformans.  Botryomycosis  is  nearly  always  a  wound 
infection.  It  may  originate  at  any  break  in  the  surface  made  by  the 
harness,  and  it  frequently  begins  in  castration  wounds.  The  process 
extends  from  the  cutaneous  and  subcutaneous  tissue  into  the  lymph 
glands  and  muscles,  where  it  may  lead  to  a  botryomycotic  myositis. 
In  castration  wounds  the  botryomycotic  masses  along  the  seminal 
cords  sometimes  assume  a  large  size  and  a  mushroom  shape.  Botry- 
omycotic tumors  of  the  chest  and  shoulders  of  the  horse  weighing 
from  fifty  to  one  hundred  pounds  have  been  reported.  Tumors  of 
smaller  size  have  also  been  found  at  the  lips,  conchse  of  the  ears, 
mammary  glands,  anus,  tail,  etc. 

Morphology  and  Staining  Properties. — The  zoogleal  masses  found  in 
the  pus  and  the  tissues  can  be  seen  with  the  naked  eye  as  pinhead- 
sized,  yellowish-white  bodies.  When  examined  microscopically  they 
appear  mulberry-shaped,  and  are  found  to  consist  of  large,  densely 
crowded  cocci.  They  are  contained  in  and  surrounded  by  a  mass  of 
protoplasm,  and  for  this  reason  are  known  as  zoogleal  masses.  The 
protoplasmic  envelope  or  capsule  is  the  thicker  the  larger  the  colony 
of  cocci.  The  individual  cocci  are  comparatively  large,  i.  e.,  1  to  1.5 
micra  in  diameter.  They  stain  best  with  anilin-water-gentian  violet. 

Cultural  and  Biologic  Properties. — The  Botryococcus  ascoformans 
can  be  easily  cultivated  on  gelatin  and  potatoes,  less  easily  on  agar. 
It  liquefies  gelatin.  The  artificial  cultures  never  show  the  zoogleal 
masses,  which  are  seen  in  the  infected  tissues  and  pus.  In  pure 
cultures  the  organism  closely  resembles  the  Staphylococcus  pyogenes 
aureus,  but  the  preponderance  of  evidence  is  that  the  botryococcus  is  a 
distinct  organism  and  not  a  variety.  While  it  is  true  that  cultures  of 
the  botryococcus,  when  inoculated  into  a  horse,  may  produce  either 
a  simple  suppuration  or  a  botryomycosis,  injections  of  the  Staphy- 
lococcus pyogenes  aureus  into  any  animal  have  never  been  known 
to  produce  a  botryomycosis. 

Experimental  Infection. — Guinea-pigs  after  inoculation  with  the 
Botryococcus  ascoformans  die  from  septicemia.  In  sheep  and  goats 
subcutaneous  infection  produces  an  edema,  sometimes  with  necrosis 
of  the  skin,  and  occasionally  followed  by  death.  Kitt  reports  that 
pigeons  and  ducks  die  after  inoculations. 


PYOGENIC  BACTERIA  OF  CATTLE. 

Suppuration  in  cattle  in  a  majority  of  cases  is  not  due  to  the  common 
staphylococci  so  frequently  found  in  man  and  the  horse,  but  to  the 
following  special  bacteria: 


PATHOLOGIC  LESIONS  207 

Streptococcus  pyogenes  bovis. 

Staphylococcus  pyogenes  bovis. 

Bacillus  pyogenes  bovis. 

Bacillus  liquefaciens  pyogenes  bovis. 

Bacillus  crassus  pyogenes  bovis. 

Streptococcus  Pyogenes  Bovis. — This  organism  appears  as  small  cocci 
arranged  in  long  chains;  they  grow  particularly  long  in  nutrient 
bouillon.  It  does  not  liquefy  gelatin,  nor  grow  on  potatoes.  Bouillon 
first  becomes  cloudy,  later  a  scanty  sediment  forms  on  the  bottom  and 
the  fluid  again  becomes  clear.  It  is  not  pathogenic  to  guinea-pigs 
and  rabbits. 

FIG.  115 


Streptococcus  pyogenes   bovis,  pure  culture  obtained  from  the  uterus  of   a  cow.      X  1000. 
(From  preparation  of  Dr.  L.  E.  Day.) 

The  Staphylococcus  Pyogenes  Bovis. — The  organism  is  smaller  than 
the  Staphylococcus  pyogenes  aureus  of  man  and  the  horse.  It 
does  not  grow  well  in  artificial  cultures,  and  soon  dies  out.  It  does 
not  liquefy  gelatin,  while  the  Staphylococcus  pyogenes  aureus  does. 
Cultures  on  artificial  media  show  a  very  low  degree  of  virulency. 


BACILLUS  PYELONEPHRITIDIS  BOVIS. 

This  bacillus  is  an  important  pus  producer  in  cattle,  frequently 
causing  a  pyelonephritis,  known  as  pyelonephritis  bacillosa  bovum. 
The  disease  is  generally  observed  in  cows  shortly  after  parturition, 
particularly  in  cases  of  retention  of  the  placenta.  Occasionally  pyelo- 
nephritis in  cows  is  also  caused  by  the  common  Staphylococcus  and 
by  the  Bacillus  pyocyaneus. 

Pathologic  Lesions. — Pyelonephritis  bacillosa  bovum  is  an  inflam- 
matory, purulent,  or  diphtheritic  necrotic  process,  characterized  by 
great  enlargement  of  one  or  both  kidneys,  with  enlargement  of  the 


208  PYOGENIC  BACTERIA  IN  DOMESTIC  ANIMALS 

pelves  and  ureters.  The  latter  contain  a  dirty  grayish  or  brownish 
purulent,  bloody  fluid.  The  urine  found  in  the  bladder  is  likewise 
purulent  and  hemorrhagic.  The  mucosa  of  the  pelvis,  ureter,  and 
bladder  are  covered  with  a  thick  tenacious  pus  and  often  with  necrotic 
diphtheritic  masses.  The  disease  is  generally  chronic,  rarely  acute, 
and  always  terminates  fatally. 

Morphology  and  Staining  Properties. — The  specific  bacilli  are  from 
2  to  3.8  micra  long  and  from  0.6  to  0.7  of  a  micron  wide.  They  are 
slender,  generally  slightly  curved,  non-motile,  and  do  not  form  spores. 
They  stain  with  the  watery  basic  anilin  stains  and  are  Gram  positive. 
They  sometimes  show  polar  granules.  They  may  be  club-shaped,  and 
occasionally  form  branches. 

Cultural  and  Biologic  Properties. — They  are  strictly  aerobic  and  will 
not  grow  on  artificial  culture  media  in  the  absence  of  oxygen.  They 
grow  readily  on  agar  and  -blood  serum  at  room  temperature,  better 
in  the  incubator  at  37°  C.,  and  form  small  grayish- white  punctate 
colonies  with  a  sharp  margin.  In  bouillon  they  form  a  fine  sediment 
after  two  days,  the  supernatant  fluid  remaining  clear.  They  grow 
neither  in  milk  nor  on  potatoes,  and  generally  not  in  gelatin.  The 
cultures  have  a  tendency  to  die  out  quickly. 

The  bacillus  is  not  pathogenic  to  man,  but  it  sometimes  leads  to 
suppuration  when  injected  into  mice  and  guinea-pigs.  Intravenous 
injection  in  cattle  after  ligation  of  the  ureter  produces  a  typical  attack 
of  pyelonephritis  bo  vis.  The  natural  mode  of  infection  in  cows  is 
through  the  genito-urinary  tract  after  parturition.  The  organism 
may  also  enter  the  kidneys  through  the  blood  current  (hematogenous 
infection). 

BACILLUS  PT06ENES  SUIS. 

Occurrence  and  Pathogenesis. — Suppuration  in  hogs  is  generally 
caused  by  a  specific  microorganism  known  as  the  Bacillus  pyogenes 
suis.  Inflammatory  suppurative  processes,  generally  confined  to  the 
serous  membranes,  particularly  the  pleura,  pericardium,  and  peri- 
toneum, are  frequently  seen  in  hogs  after  they  are  slaughtered.  The 
membranes  are  thickened  by  inflammatory  connective  tissue,  and 
excrescences  in  the  form  of  more  or  less  spherical  or  elliptical  masses 
project  beyond  their  surface.  These  masses  are,  as  a  matter  of  fact, 
small  abscesses  surrounded  by  a  capsule  of  tough,  fibrous,  connective 
tissue.  If  incised,  the  abscesses  discharge  a  thick,  tenacious,  yellowish- 
green  or  greenish  non-fetid  pus.  In  advanced  cases  abscesses  are 
also  found  in  the  lymph  nodes  of  the  thorax,  head,  and  muscles. 
Occasionally  they  are  found  over  the  entire  body,  in  all  the  internal 
organs. 

Morphology  and  Staining  Properties. — The  microscopic  examination 
of  pus  from  hogs  shows  a  short,  slender,  non-motile  bacillus,  often 
present  in  very  large  numbers.  It  stains  best  with  anilin-water 
gentian  violet  or  carbol-fuchsin;  it  is  Gram  negative  and  is  not  acid 


QUESTIONS  209 

fast.  In  old  abscesses  the  bacilli  present  involution  forms  and  stain 
poorly. 

Cultural  and  Biologic  Properties. — The  Bacillus  pyogenes  suis  grows 
best  on  coagulated  blood  serum  at  blood  temperature  in  the  incubator. 
It  also  grows  on  agar,  in  gelatin  stick  cultures,  and  in  bouillon.  The 
latter  remains  clear,  or  becomes  very  slightly  cloudy.  A  whitish 
sediment  collects  at  the  bottom  of  the  tube.  On  blood  serum  whitish, 
delicate,  dry,  punctate  colonies,  which  liquefy  the  culture  soil  slightly, 
develop  after  several  days.  On  potatoes  a  similar  growth  occurs. 
No  gas  is  formed  in  glucose  agar. 

Experimental  Inoculation. — The  Bacillus  pyogenes  suis  is  pathogenic 
to  rabbits  and  mice  when  injected  into  the  peritoneal  cavity.  It  pro- 
duces a  purulent  peritonitis,  with  a  tendency  to  encapsulated  abscess 
formation,  resembling  that  occurring  in  the  hog.  In  hogs  the  natural 
mode  of  infection  is  by  inhalation  or  through  wounds,  such  as 
castration  wounds,  injuries  in  the  mouth,  umbilical  cord,  etc. 

QUESTIONS. 

1.  What  organism  is  most  commonly  the  cause  of  suppuration  in  horses? 

2.  What  other  organism  causing  suppuration  in  man  causes  a  similar  process 
in  the  horse? 

3.  What  organism  causes  strangles  in  the  horse?    Under  what  other  names 
is  this  infection  known? 

4.  Where  is  the  Streptococcus  equi  found  in  strangles  and  what  other  equine 
diseases  does  it  sometimes  cause? 

5.  Describe  the  morphology  of  the  Streptococcus  equi. 

6.  What  are  its  staining  properties? 

7.  Describe  a  bouillon  culture  of  the  Streptococcus  equi. 

8.  Also  a  growth  on  agar,  gelatin,  and  blood  serum. 

9.  What  animals  are  susceptible  to  infection  with  the  Streptococcus  equi? 

10.  What  bacteria  have  been  found  in  the  blood  of  horses  dead  from  Morbus 
maculosus? 

11.  Describe  the  organism  found  bySchiitz  in  contagious  pleuropneumonia  of 
horses. 

12.  Describe  the  organisms  found  as  the  cause  of  apoplectiform  septicemia  in 
chickens. 

13.  What  kind  of  disease  is  botryomycosis?    What  is  its  etiologic  factor? 

14.  Why  is  botryomycosis  in  some  of  its  features  like  tumor  formation? 

15.  Describe  the  seat  and  appearance  of  botryomycotic  lesions. 

16.  Describe  the  Botryococcus  ascoformans  as  seen  in  pus  both  with  the  naked 
eye  and  with  the  microscope. 

17.  What  is  a  zoogleal  mass?  how  is  its  formation  brought  about?    How  does 
it  look  in  a  pure  culture  of  Botryococcus  ascoformans? 

18.  How  does  the  organism  look  in  pure  culture  on  agar? 

19.  For  what  animals  is  the  organism  pathogenic? 

20.  Under  what  conditions  does  the  Staphylococcus  pyogenes  aureus  produce 
botryomycosis  ? 

21.  Name  the  bacteria  which  are  generally  the  causes  of  suppuration  in  cattle. 

22.  What  is  generally  the  cause  of  pyelonephritis  in  cattle?     Describe  the 
organism  and  the  pathogenic  changes  which  it  produces. 

23.  What  are  the  cultural  properties  of  this  organism? 

24.  What  is  a  hematogenous  infection? 

25.  What  organism  generally  produces  suppuration  in  hogs?     Describe  its 
morphology. 

26.  Describe  the  abscesses  due  to  this  organism. 

27.  Describe  the  cultural  properties  of  the  Bacillus  pyogenes  suis. 

28.  What  animals  are  susceptible  to  it? 

14 


CHAPTEE    XVIII. 

BACTERIA   PRODUCING   DIPHTHERITIC    INFLAMMATIONS- 
BACILLUS  DIPHTHERIA— BACILLUS  NECROPHORUS— 
BACILLUS  DIPHTHERIA  AVIUM. 

A  NUMBER  of  anatomical  types  of  inflammation,  such  as  serous, 
fibrinous,  purulent,  hemorrhagic,  and  diphtheritic,  are  distinguished 
in  pathology.  The  microorganisms  most  commonly  causing  purulent 
inflammations  have  been  described  in  the  two  preceding  chapters. 
Some  of  the  most  important  organisms  causing  diphtheritic  inflam- 
mations will  now  be  considered.  A  diphtheritic  inflammation  is  one 
marked  by  extensive  necrosis,  either  due  in  the  beginning  to  chemical 
or  physical  influences  (acids,  alkalies,  heat,  cold),  or  resulting,  at  an 
early  stage,  from  the  toxins  of  the  pathogenic  invading  and  multi- 
plying organisms.  In  either  case  an  area  of  tissue  containing  numerous 
necrotic  cells  is  formed;  generally  on  the  surface  adjacent  to  the 
necrotic  zone  there  is  a  hyperemic  one  with  dilated  vessels,  with  an 
abundant  transudate  and  numerous  migrated  leukocytes.  Diphtheritic 
inflammations  tend  to  form  pseudomembranes,  which  are  grayish 
white,  yellowish  white,  dirty  gray,  or  if  mixed  with  a  great  number  of 
erythrocytes,  dark  gray  or  dirty  brown  in  color.  These  membranes, 
when  removed  artificially,  or  when  shed  in  the  natural  course  of  the 
necrosis,  leave  a  raw,  ulcerated,  often  bleeding  surface. 


BACILLUS  DIPHTHERIA. 

Occurrence  and  Pathogenesis. — Diphtheritic  inflammations  occur  in 
man  and  the  domestic  animals.  In  man  diphtheria  is  generally  a 
disease  of  the  tonsils,  pharynx,  and  larynx,  although  it  may  also  occur 
in  accidental  or  operative  wounds.  It  is  caused  by  the  bacillus  of 
diphtheria. 

Morphology. — This  bacillus,  as  found  in. recent  diphtheritic  inflam- 
mations or  obtained  from  pure  cultures  raised  on  Loeffler's  blood- 
serum  mixture,  shows  the  following  morphologic  features:  The 
bacillus  varies  considerably  in  length  from  1  to  6  micra;  the  majority 
being  about  3  micra.  It  is  from  0.3  to  0.8  of  a  micron  thick. 
In  shape  it  is  frequently  slightly  curved,  and  in  very  rare  cases 
cylindrical.  It  is  generally  thickened  at  one  end  or  more  rarely 
at  both  ends,  making  it  either  club-shaped  or  dumb-bell-shaped. 
When  it  forms  chains  they  are  always  short.  On  division  and  multi- 
plication the  bacilli  have  a  tendency  to  separate  immediately  at  the 


BACILLUS  DIPHTHERIA 


211 


constricting  line  of  division  and  are,  for  this  reason,  frequently  found 
in  groups  of\parallel  rows.  These  groups  somewhat  resemble  palli- 
sades,  and,  hence,  a  pallisade  arrangement  of  the  diphtheria  bacilli 
from  pure  cultures  is  spoken  of.  The  bacillus  does  not  form  spores  and 
is  not  motile. 

Cultural  and  Staining  Properties. — The  diphtheria  bacillus  or  Klebs- 
Loeffler1  bacillus  is  best  stained  with  Loeffler's  alkaline  methylene 
blue.  When  derived  from  a  young  blood-serum  culture  (eighteen  to 
twenty-fours  hours'  incubation)  it  generally  shows  very  typical  staining 
properties.  It  does  not  take  the  dye  uniformly,  so  that  stained  spaces 
alternate  with  unstained  spaces.  The  stain  is  generally  taken  at 
either  end  and  by  a  segment  in  the  middle.  This  causes  diphtheria 
bacilli  frequently  to  appear  to  the  beginner  like  short  chains  of  strepto- 
cocci, but  a  more  careful  examination  will  show  them  to  be  unequally 


FIG.  116 


FIG.  117 


Pseudodiphtheria  bacilli.    (Park.) 


One  of  the  very  characteristic  forms  of 
diphtheria  bacilli  from  blood-serum  cul- 
tures, showing  clubbed  ends  and  irreg- 
ular stain.  X  1100  diameters.  Stain, 
methylene  blue.  (Park.) 


stained  bacilli.  When  grown  on  agar  they  do  not  show  this  typical 
behavior,  but  become  much  shorter  and  stain  more  uniformly,  resem- 
bling then  more  closely  the  non-pathogenic  bacillus  known  as  the 
pseudodiphtheria  bacillus.  On  this  account  Loeffler's  blood-serum 
mixture2  should  always  be  used  in  making  cultural  inoculations  from 
suspected  diphtheria  cases.  The  Bacillus  diphtheria?  retains  Gram's 
stain.  It  also  grows  on  agar,  on  bouillon,  and  in  milk.  A  twenty-four 
hour  culture  on  blood-serum  mixture  or  agar  shows  comparatively 
small,  grayish- white,  granular,  moderately  dry  or  slightly  moist  colonies. 
The  bacillus  grows  in  the  presence  or  absence  of  oxygen,  best  at 
blood  temperature.  It  does  not  liquefy  gelatin  and  does  not  grow  well 
on  it.  It  forms  gas  in  the  presence  of  glucose.  Pure  cultures  are 
generally  obtained  by  first  inoculating  the  blood-serum  mixture, 


1  Named  Klebs-Loeffler,  after  its  discoverers. 

2  The  formula  for  Loeffler's  blood-serum  mixture  is  given  on  p.  133. 


212     BACTERIA  PRODUCING  DIPHTHERITIC  INFLAMMATIONS 

keeping  it  in  the  incubator  for  eighteen  hours  and  then  pouring  plates 
with  glycerin  agar.  In  the  cultivation  of  larger  masses  of  the  bacilli 
for  the  production  of  the  toxin,  bouillon  is  generally  employed.  Some 
strains  of  bacilli  grow  readily  and  abundantly  on  it,  others  only  feebly. 
The  bouillon  should  be  slightly  alkaline  to  litmus.  Frequently  the 
growing  and  multiplying  bacilli,  after  twenty-four  to  forty-eight 
hours,  produce  a  diffuse  cloudiness  in  the  bouillon  and  form  a  film 
or  pellicle  on  its  surface. 

Toxin  Formation. — The  diphtheria  bacillus  forms  a  soluble,  very 
poisonous  toxin,  which  when  inoculated  into  a  susceptible  animal, 
produces  all  the  general  symptoms  of  the  disease. 

Diphtheria  Antitoxin. — This  is  prepared  by  the  systematic  injection 
of  diphtheria  toxin  and  subsequently  cultures  of  diphtheria  bacilli 
into  a  perfectly  healthy  horse.  The  technique  and  details  are  almost 
identical  with  those  employed  in  the  preparation  of  tetanus  antitoxin, 
and  are  fully  described  in  the  chapter  on  the  Bacillus  tetani. 

Animals  Susceptible. — Young  cats  in  houses  where  diphtheria  occurs 
among  children,  frequently  contract  the  disease.  Guinea-pigs,  young 
cats,  young  rabbits,  and  other  animals  can  easily  be  infected  experi- 
mentally. Many  animals  are  susceptible  to  experimental  intraperi- 
toneal  injection  with  diphtheria  toxin. 

In  making  a  diagnosis  of  diphtheria  the  possibility  of  the  presence 
of  the  pseudodiphtheria  bacillus  must  always  be  considered.  As  the 
latter  cannot  always  be  distinguished  morphologically  from  the  true 
Bacillus  diphtherise,  animal  experiments  are  sometimes  necessary  to 
decide  the  question. 


BACILLUS  NECROPHORUS. 

The  Bacillus  necrophorus  was  first  found  by  Loeffler  in  diphtheria 
of  calves  and  called  by  him  Bacillus  diphtherise  vitulorum  (Latin, 
vitulus,  a  calf).  It  is  also  known  as  Streptothrix  necrophora,  Bacillus 
necroseus,  Streptothrix  cuniculi  (Latin,  cuniculus,  a  rabbit). 

Occurrence  and  Pathogenesis. — It  is  a  very  common  cause  of  diph- 
theritic, necrotic  inflammations  among  domestic  animals.  Ostertag, 
quoting  Bang,  enumerates  the  following  pathologic  conditions  in 
which  it  has  been  found: 

Diphtheria  of  calves. 

Furunculosis  of  cattle. 

Dry  gangrene  of  the  udder  of  cows. 

Multiple  necrotic  foci  in  the  liver  of  cattle. 

Multiple  abscess  in  the  liver  of  cattle. 

Diphtheritis  of  the  uterus  and  vagina  of  cows. 

Diphtheritic  necrosis  of  the  small  intestines  of  calves. 

Embolic  pulmonary  necrosis  in  cattle. 

Embolic  myocardial  necrosis  in  cattle. 


PLATE  IV 


V  v.)  M  *»    v  s«T 
i    *     /«     .  •     '^  /^ 

^i  ^L  £     ^••v^ 


Section  of  the  Lung  of  a  Horse.    Bacillus  Neerophorus  Infectior 


BACILLUS  NECROPHORUS  213 

Wound  necrosis  in  cattle. 

Necrosis  of  the  hoof  cartilages  of  the  horse. 

Diphtheria  of  the  intestines  in  horses. 

Diphtheritic  necrosis  in  the  mouth,  nose,  and  intestines  of  hogs. 

Multiple  necrosis  in  the  liver  of  sheep. 

Multiple  necrosis  in  the  liver  of  mules. 

Diphtheria  of  Calves. — In  the  diphtheria  of  calves  the  mouth  and 
pharynx  show  diphtheritic  pseudomembranes.  In  advanced  and  fatal 
cases  diphtheritic  necrosis  is  also  found  in  the  intestines  and  lungs. 
The  affected  areas  appear  as  yellowish,  irregular,  necrotic  patches, 
covered  by  diphtheritic  membranes. 

Multiple  Liver  Abscesses  in  Cattle. — Multiple  necrotic  foci  or  mul- 
tiple abscesses,  sometimes  as  large  as  an  apple  and  even  larger,  are 
frequently  found  in  the  livers  of  cattle.  They  are  surrounded  by  a 
tough,  fibrous  connective-tissue  capsule  and  contain  a  very  tenacious, 
thick,  generally  greenish  non-fetid  pus,  in  which  there  is  much 
granular  necrotic  material.  In  it  bacilli  and  filaments  are  found 
which  by  culture  and  inoculation  experiments  can  be  identified  as 
the  Bacillus  necrophorus.  Occasionally  the  bacillus  is  associated 
with  the  Bacillus  pyogenes  bovis. 

Furunculosis  of  the  Horse's  Hoof  with  Necrosis  of  the  Cartilages. — 
Necrophorus  infection  of  the  hoof  of  the  horse  is  very  common  in 
certain  seasons  and  localities.  It  appears  particularly  in  winter  and 
was  very  prevalent  in  1909  and  1910.  The  infection  causes  progressive 
necrosis  of  the  cartilages  and  often  leads  to  changes  which  permanently 
damage  the  locomotion  of  the  animal.  In  spite  of  antiseptic  treatment 
and  operative  procedures  the  process  tends  to  spread.  Sometimes 
it  also  produces  a  general  infection  with  the  formation  of  multiple 
metastatic  bacterial  emboli  in  the  liver  and  lungs.  The  author  has 
seen  the  case  of  a  horse  which  died  of  a  general  necrophorus  septico- 
pyemia  with  the  formation  of  metastases  in  the  internal  organs.  The 
lung  presented  the  picture  of  a  lobular  or  bronchopneumonia  and  in 
the  consolidated  areas  the  Bacillus  necrophorus  was  found  in  enor- 
mous numbers. 

Foot-rot  and  Lip-and-leg  Disease  of  Sheep. — Mohler  and  Washburn 
have  fully  established  the  Bacillus  necrophorus  as  the  etiologic  factor 
of  this  disease,  which  is  now  widespread  in  the  United  States  and  of 
considerable  importance.  They  describe  the  symptoms,  lesions,  and 
course  as  follows: 

'  The  first  evidence  of  an  attack  of  foot-rot  to  attract  the  attention 
is  a  slight  lameness,  which  rapidly  becomes  more  marked.  Previous 
to  this,  however,  there  has  appeared  a  moist  area  just  above  the 
horny  part  of  the  cleft  of  the  foot,  and  this  has  gradually  reddened 
and  assumed  a  feverish,  inflamed  appearance.  It  may  first  become 
visible  either  at  the  front  or  back  part  of  the  cleft,  but  usually  the 
erosions  make  their  first  appearance  at  the  heel.  The  inflammation 
rapidly  penetrates  beneath  the  horny  tissue,  while  from  the  ulcerous 


214     BACTERIA  PRODUCING  DIPHTHERITIC  INFLAMMATIONS 

opening  there  exudes  a  thin,  purulent  fluid.  The  lameness  increases 
and  the  region  of  the  foot  above  the  hoof  becomes  swollen  and  warm  to 
the  touch.  The  exudates  from  the  erosions  contain  pus  cells,  bits 
of  destroyed  tissues  of  the  foot,  and  bacteria.  It  possesses  an  odor 
pungent  and  disagreeable,  but  at  the  same  time  very  characteristic 
This  odor  is  so  pathognomonic  of  the  disease  that  it  would  reveal  the 
presence  of  infected  sheep  to  one  familiar  with  the  character  of  the 
infection,  even  before  noticing  the  animals. 

"  The  invasion  of  the  necrotic  process  may  continue  until  ligaments, 
tendons,  and  even  the  bones  are  attacked,  but  before  this  final  stage 
is  reached  nature  will  attempt  to  repair  the  damage. 

FIG.  118 


_ 


1 


Leg-and-lip  disease  in  sheep.     Infection  with  Bacillus  necrophorus.     (Dolan.) 

"The  hoof  of  a  sheep  suffering  from  a  chronic  case  of  foot-rot 
grows  out  rapidly  and  becomes  very  hard.  It  will  often  be  found 
with  the  toes  so  thickened  and  lengthened  that  the  front  part  of  the 
foot  is  raised  above  its  natural  incline  and  the  tendons  at  the  heel 
are  subjected  to  additional  strain,  all  of  which  tends  to  increase  the 
lameness  and  the  awkwardness  in  gait  of  the  victim.  These  thickened 
and  elongated  toes  will  frequently  be  seen  to  have  attained  an  added 
length  of  three  or  even  four  inches,  and  they  curl  up  like  sled  runners, 
greatly  interfering  with  the  progression  of  the  animal. 

"  The  course  of  this  disease  is  slow  and  protracted,  usually  starting 
with  one  foot  and  subsequently  involving  one  or  more  of  the  others. 
During  this  interval  it  will  probably  spread  to  the  feet  of  other  sheep, 


BACILLUS  NECROPHORUS 


215 


and  in  this  way  the  disease  may  remain  for  several  months  in  each 
member  of  the  flock,  and  for  eight  or  ten  months  in  the  flock  itself. 


FIG.   119 


Leg-and-lip  disease  in  sheep.     Infection  with  Bacillus  necrophorus.      (Dolan.) 

When  the  ulcerous  processes  have  become  advanced  and  aggravated, 
fever  develops,  the  appetite  is  lost,  and  the  animal  grows  so  emaciated 
that  death  intervenes.  In  some  cases  that  are  left  untreated  recovery 


FIG.  120 


Leg-and-lip  disease  in  sheep.     Infection  with  Bacillus  necrophorus.     (Dolan.) 

may  follow  slowly,  but  there  is  usually  either  a  dense  fungoid  growth 
between  the  claws,  a  stiffening  of  the  joints  of  the  ankle,  or  a  long 


216     BACTERIA  PRODUCING  DIPHTHERITIC  INFLAMMATIONS 


FIG.  121 


fissured  and  misshapen  hoof.  When  treatment  is  properly  applied 
in  the  early  stages  of  the  disease  it  is  usually  cured  within  ten  days. 
It  is  very  rare  for  death  to  occur  as  a  result  of  foot-rot,  although  in 
very  virulent  outbreaks  involving  three  or  four  feet  of  each  sheep 
the  affection  may  terminate  fatally  within  two  or  three  months." 

Secondary  necrophorus  ulcerations  frequently  occur  on  the  lips  of 
sheep  as  a  result  of  infection  from  licking  the  ulcerations  on  the  feet. 
The  disease  is  then  termed  lip-and-leg  ulcer- 
ation.  The  contagion  may  also  affect  the 
male  and  female  genitalia,  in  which  case  it  is 
known  as  necrotic  venereal  disease  of  sheep. 
Morphology  and  Staining  Properties. — The 
organism,  when  found  in  pus  and  necrotic 
material,  appears  not  only  as  a  bacillus,  but 
also  in  long  filaments,  which  give  it  the 
character  of  a  streptothrix.  The  filament 
sometimes  break  up  into  very  short  segments, 
and  may  then  appear  like  a  true  streptothrix. 
The  filaments  are  from  80  to  100  micra  in 
length.  The  organism  is  best  stained  with 
Loeffler's  methylene  blue,  carbol  fuchsin,  or 
carbol  thionin.  In  stained  specimens  of  the 
filamentous  type,  unstained  spaces  often 
alternate  with  short,  stained,  cylindrical  rods, 
causing  the  filament  to  look  somewhat  like 
a  stepladder.  In  sections  of  tissues  the 
bacilli,  threads,  and  filaments  show  a  radial 
arrangement,  and  they  are  seen  most  clearly 
and  in  greatest  numbers  at  the  boundary 
zone  between  the  necrotic  and  the  hyper- 
emic  tissues.  Branched  forms  frequently 
occur.  It  does  not  form  spores  and  the 
long  filaments  are  not  motile,  but  the  short 
bacilli  when  first  seen  in  pus,  exhibit  a 
slight  motion.  Flagella,  however,  have  not 
been  demonstrated,  and  the  motility  is  soon 
lost. 

Cultural  and  Biologic  Properties. — The  or- 
ganism is  strictly  anaerobic  and  will  not  grow 
in  the  presence  of  oxygen,  It  grows  best  on 
blood  serum  or  a  mixture  of  agar  and  blood  serum  at  30°  to  40°  C.  It 
also  develops  on  agar  and  gelatin.  In  stab  cultures  in  high  blood-serum 
tubes  small  whitish  points  appear  along  the  stab  after  twenty-four  to 
forty-eight  hours.  When  a  maximum  of  growth  has  been  reached, 
which  takes  six  or  eight  days,  an  opaque,  grayish-white,  cylindrical 
mass,  surrounded  by  a  transparent  zone  of  small  individual  colonies 
appears  along  the  stab.  Its  great  susceptibility  to  oxygen  and  its 


Leg-and-lip  disease  in 
sheep.  Infection  with  Bacil- 
lus necrophorus.  (Dolan.) 


PLATE  V 


Cover-glass   Preparation  from  Pure  Culture  of  Bacillus 
Neerophorus.     (Mohler  and  Washburn). 


BACILLUS  NECROPHOROUS 
FIG.  122  FIG.  123 


217 


Bouillon-agar  culture  (first  dilution)  of 
Bacillus  necrophorus,  showing  twenty-four- 
hour  growth,  with  numerous  small  gas- 
bubbles,  but  the  colonies  have  not  developed 
sufficiently  to  become  visible.  (Mohler  and 
Washburn.) 


Seven-day-old  bouillon-agar  culture  of  this 
organism  of'the  fourth  dilution.  The  isolated 
colonies  are  characteristic  in  that  their  gray- 
ish centres  are  surrounded  by  fuzzy  white 
areas,  not  unlike  the  strands  of  loose,  fleecy 
cotton.  (Mohler  and  Washburn.) 


FIG.  124 


Single  colonies  of  the  necrosis  bacillus,  showing  filamentous  character  of   the  growth 
(enlarged  about  seven  diameters).     (Mohler  and  Washburn.) 


218     BACTERIA  PRODUCING  DIPHTHERITIC  INFLAMMATIONS 

strictly  anaerobic  properties  are  shown  by  the  fact  that  the  culture 
never  reaches  the  surface  of  the  serum  medium.  In  the  condensed 
water  of  serum  agar  the  growth  forms  a  grayish-white  film  or  pellicle 
and  a  sediment  of  the  same  color.  When  grown  in  bouillon  or 
milk  a  smell  of  cheese  is  given  off;  the  fluid  media  then  give  the 
indol  reaction.  The  organism  is  frequently  found  in  the  intestines  of 
herbivorous  animals,  particularly  the  hog.  Pure  cultures  cannot,  as 
a  rule,  be  obtained  directly  from  the  lesions,  because  the  organism  is 
rarely  present  in  that  condition,  but  is  usually  mixed  with  other  patho- 
genic or  saprophytic  organisms.  The*  method  of  procedure  consists 
in  inoculating  the  material  subcutaneously  into  a  rabbit.  A  very 
hard  inflammatory  induration  is  then  formed  at  the  site  of  the  in- 
jection, where  the  tissues  subsequently  became  caseous  and  necrotic. 
The  necrophorus  bacillus  at  first  multiplies  locally,  and  afterward 
enters  the  general  lymph  and  blood  circulation,  leading  to  the  forma- 
tion of  metastases  in  the  internal  organs,  particularly  the  lungs  and 
the  liver.  The  rabbit  generally  dies  in  less  than  two  weeks,  and 
anaerobic  cultures  can  be  made  from  the  internal  metastases,  which 
usually  contain  the  organism  in  pure  culture.  The  disease  also  occurs 
spontaneously  in  rabbits,  starting  as  an  infection  of  the  face. 


BACILLUS  DIPHTHERIA  AVIUM. 

Occurrence  and  Pathogenesis. — A  highly  contagious  disease  char- 
acterized anatomically  by  diphtheritic  inflammations  of  the  mucous 
membranes  of  the  head  occurs  among  pigeons,  domestic  and  prairie 
chickens,  turkeys,  and  other  fowl.  The  diphtheritic  pseudomem- 
branes  are  found  in  the  mouth,  pharynx  and  nose,  and  its  accessory 
cavities,  less  frequently  in  the  larynx,  trachea,  bronchi,  and  intestines. 
In  the  latter  the  cecal  pouches  are  sometimes  completely  filled 
with  the  diphtheritic  membranes.  The  cause  of  the  disease  is  a 
short  bacillus  which  was  first  found  by  Loeffler  in  diphtheria  of 
pigeons.  It  was,  accordingly,  called  Bacillus  diphtherise  colum- 
barum.  Later,  Moore  and  others  found  the  same  bacillus  in 
chicken  diphtheria,  and  it  is  now  generally  known  under  the  more 
general  term  of  the  bacillus  of  bird  diphtheria  (Bacillus  diphtherise 
avium). 

Morphology  and  Staining  Properties. — The  organism  is  a  short  bacillus, 
not  motile,  and  does  not  form  spores.  It  stains  in  a  bipolar  manner 
with  the  watery  anilin  stains,  and  is  Gram  negative. 

Cultural  Properties. — It  grows  both  in  the  presence  and  absence  of 
oxygen.  On  gelatin,  which  is  not  liquefied,  a  grayish-white  transparent 
growth  composed  of  finely  granular  colonies  appears  first,  later  the 
growth  becomes  white  and  opaque.  On  agar  the  growth  is  similar, 
but  first  slightly  bluish,  then  white  and  opaque,  as  on  gelatin.  Bouillon 
becomes  first  uniformly  cloudy,  later  a  somewhat  transparent  white 


QUESTIONS  219 

sediment  is  formed.  On  potatoes  either  a  yellowish-white  or  a  grayish- 
white  growth  is  formed,  but  occasionally  the  organism  refuses  to 
develop  on  this  culture  soil.  The  organism  is  also  pathogenic  for 
mice  and  rabbits. 

QUESTIONS. 

1.  What  are  the  characteristic  pathologic  changes  in  diphtheritic  inflam- 
mations? 

2.  What  is  the  cause  of  diphtheria  in  man? 

3.  Describe  the  organism  causing  the  disease. 

4.  Describe  its  staining  properties  when  stained  with  Loeffler's    alkaline 
methylene  blue.    Give  the  formula  for  this  stain. 

5.  Give  the  formula  for  the  preparation  of  Loeffler's  blood-serum  mixture. 

6.  Describe  the  growth  of  the  Bacillus  diphtherias  on  this  culture  medium. 

7.  What  non-pathogenic  bacillus  in  cultures  and  stained  microscopic  speci- 
mens looks  very  much  like  the  Bacillus  diphtherias? 

8.  How  does  this  bacillus  act  toward  oxygen,  glucose,  gelatin? 

9.  What  kind  of  toxins  are  formed  by  the  diphtheria  bacillus? 

10.  What  domestic  animals  contract  diphtheria  spontaneously? 

11.  Name  a  number  of  animal  diseases  due  to  the  Bacillus  necrophorus. 

12.  What  other  names  have  been  given  to  the  Bacillus  necrophorus? 

13.  What  is  the  arrangement  of  this  bacillus  in  infected  tissues? 

14.  What  are  the  cultural  and  biologic  properties  of  this  organism? 

15.  How  are  pure  cultures  obtained  from  pathologic  material  containing  the 
Bacillus  necrophorus  and  other  bacteria? 

16.  Describe  the  pathologic  lesions  caused  by  the  organism  in  calves. 

17.  Give  the  description  and  common  cause  of  multiple  liver  abscesses  in 
cattle. 

18.  What  affection  of  the  horse's  hoof  is  frequently  caused  by  the  Bacillus 
necrophorus?    What  occurs  if  this  hoof  disease  leads  to  the  death  of  the  animal? 

19.  What  disease  of  sheep  is  caused  by  the  Bacillus  necrophorus? 

20.  Describe  the  pathologic  lesions  as  seen  in  the  feet  of  affected  sheep. 

21.  What  other  parts  of  the  body  of  sheep  may  become  infected  with  the 
Bacillus  necrophorus? 

22.  What  animals  are   spontaneously  infected  by  the   Bacillus  diphtheriae 
avium? 

23.  Describe  the  pathologic  lesions  produced. 

24.  Give  the  morphologic  and  cultural  properties  of  the  Bacillus  diphtheriae 
avium. 


CHAPTER    XIX. 

BACILLI  OF  THE  HEMORRHAGIC   SEPTICEMIA  GROUP— BACILLUS 
AVISEPTICUS,  BOVISEPTICUS,  OVISEPTICUS,  SUISEPTICUS, 
AND  EQUISEPTICUS— BACILLUS  OF  DOG  TYPHOID- 
PLAGUE  BACILLUS  IN  MAN  AND  ANIMALS. 

MANY  bacterial  infections,  when  they  take  a  violent,  rapidly  fatal 
course,  may  lead  to  hemorrhagic  septicemia,  that  is,  to  a  general 
infection  of  the  blood  with  extensive  multiplication  of  the  bacteria  in 
the  blood  current  and  with  the  formation  of  areas  of  hemorrhagic 
inflammation  in  the  mucous  and  serous  membranes  and  in  the  various 
internal  organs  of  the  body.  A  staphylococcus  or  a  streptococcus 
infection  may  lead  to  a  hemorrhagic  septicemia  of  this  kind,  but 
considering  the  great  number  of  infections  with  these  organisms  in 
man  and  domestic  animals  this  result  is  not  very  common. 

On  the  other  hand,  certain  bacteria,  in  a  great  majority  of  cases, 
lead  to  such  hemorrhagic  septicemias,  and  they  are  known  as  the 
group  of  bacilli  of  hemorrhagic  septicemia.  These  organisms  have 
a  number  of  common  features.  All  are  rather  small,  short  bacilli, 
with  rounded  ends,  which  do  not  form  spores,  do  not  liquefy  gelatin, 
are  non-motile  and  Gram  negative,  and  stain  in  a  peripheral  or  polar 
manner  so  that  on  first  sight  they  often  appear  like  diplococci.  Other 
bacteria,  such  as  the  anthrax  bacillus,  also  commonly  produce  a 
hemorrhagic  septicemia,  but  they  have  different  morphologic,  cul- 
tural, and  biologic  properties,  and  do  not  belong  to  this  group. 

Historical. — Rivolto  and  Semmer,  in  1878,  and  Pasteur,  in  1880, 
described  a  bacillus  of  the  type  indicated  as  the  cause  of  fowl  cholera; 
Gaffky,  in  1881,  a  similar  one  as  the  cause  of  septicemia  in  rabbits; 
Kitt,  in  1883,  one  for  the  disease  among  wild  animals  called  "Wild- 
seuche,"  by  Bollinger;  and  Loeffler,  and  later  Smith,  in  1886,  one  as 
the  cause  of  swine  plague  or  "Schweineseuche."  Hueppe,  from  his 
studies  of  the  various  bacilli  of  this  group,  concluded  that  they  were 
more  or  less  identical,  and  proposed  to  classify  them  as  the  group  of 
bacilli  of  hemorrhagic  septicemia.  It  was  subsequently  ascertained 
that  bacilli  of  this  group  are  also  the  cause  of  infectious  pleuropneu- 
monia  of  calves,  barbone  disease  of  buffaloes,  the  hemorrhagic  septi- 
cemias of  cattle,  infectious  pneumonia  of  goats,  infectious  pneumonia 
of  horses,  and  a  hemorrhagic  g astro-enteritis  of  dogs.  Ligniere  and 
Trevisans,  in  1890,  gave  the  name  of  Pasteurella  to  the  diseases  of 
this  group,  and  designated  the  bacteria  themselves  as  Pasteurelloses.1 

1  Kitt,  with  all  due  respect  for  the  genius  of  Pasteur,  severely  criticized  an  attempt  of  this 
kind  to  change  well-known  names,  which  could  only  lead  to  confusion  in  the  nomenclature  of 
diseases  and  their  causative  factors.  He  ironically  remarks  that  a  general  adoption  of  such  a 
principle  would  lead  to  names  of  diseases  and  their  bacteria  like  Kochella  (for  tuberculosis), 
Loefflerella  or  Schuetzella  (for  glanders),  Schulzerella,  Millerelose,  Smithellose,  etc. 


BACILLUS  AVISEPTICUS  221 

These  names,  however,  have  not  been  generally  adopted  and  are  only 
used  by  the  French.  It  is  preferable,  as  Hutyra  and  Marek  point 
out,  to  retain  the  name  Bacillus  bipolaris  septicus  and  to  use  as  dis- 
tinguishing designation  the  names  Bacillus  avisepticus,  bovisepticus, 
suisepticus,  etc.  Kitt  proposed  the  term  Bacillus  plurisepticus  as 
the  common  name  for  all  these  bacteria.  The  disease  which  they 
produce  in  such  a  large  variety  of  animals  he  designated  as  septiccemia 
pluriformis  or  septiccemia  polymorpha. 


BACILLUS  AVISEPTICUS  (BACILLUS  OF  FOWL  CHOLERA). 

Historical  and  Occurrence. — This  disease  of  chickens  and  other 
domestic  birds  is  also  known  as  chicken  cholera,  fowl  typhoid  (Gefliigel- 
typhoid,  German),  Pasteurelosis  avium  (cholera  des  poules,  French). 
It  is  one  of  the  diseases  due  to  bacilli  of  the  hemorrhagic  septicemia 
group,  and  has  long  attracted  the  attention  of  breeders  and  veterin- 
arians. It  was  first  described  toward  the  close  of  the  eighteenth 
century,  and  its  extremely  contagious  character  was  recognized  by 
Delafond  and  Renault  in  1851.  Perroncito,  in  1878,  first  saw  the 
causative  microorganisms  in  the  blood  of  fowls  which  had  died  from 
the  disease,  but  he  mistook  it  for  a  diplococcus,  an  excusable  error 
at  this  early  stage  of  the  study  of  pathogenic  bacteria.  Toussaint, 
in  1879,  and  Pasteur,  in  1880,  confirmed  Perroncito's  findings.  The 
two  French  investigators  also  cultivated  the  organism  in  chicken 
broth,  and  Pasteur  showed  that  it  could  be  attenuated  in  artificial 
cultures  and  afterward  used  as  a  protective  virus.  This  work  of 
Pasteur  was  the  first  of  its  kind,  and  it  became  a  fruitful  source  of 
further  attempts  in  the  preparation  of  attenuated  cultures  to  be  used 
as  protective  vaccines.  Kitt,  Ligniere,  Rivolta,  Zuern,  Celli,  Solomon, 
and  others  subsequently  studied  the  bacillus  causing  fowl  cholera, 
and  with  the  exception  of  the  bacillus  of  bubonic  plague,  it  is  at 
present  the  best-known  organism  of  the  group  of  bacilli  of  hemorrhagic 
septicemia. 

Fowl  cholera  is  very  prevalent  in  Europe  (with  the  exception  of 
Great  Britain)  and  South  Africa,  and  it  has  been  found  also  in  the 
United  States  and  Canada.  According  to  official  statistics,  48,797 
chickens,  23,573  geese,  and  9488  ducks  died  from  fowl  cholera  in 
Germany  in  1903.  These  figures  comprise  only  those  cases  in  which 
an  exact  diagnosis  was  made,  and  indicate,  of  course,  only  a  part  of 
the  actual  loss. 

Pathologic  Lesions. — Birds  which  have  died  from  fowl  cholera  show 
numerous  hemorrhages  into  the  mucous  and  serous  membranes. 
Among  the  serous  membranes  the  epicardium,  or  external  lining 
membrane  of  the  heart,  shows  particularly  numerous  hemorrhagic 
spots.  This  change  is  most  marked  in  geese  and  ducks,  less  in 
chickens.  A  serous  or  fibrinous  pericarditis,  a  hemorrhagic  enteritis 


222     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

(inflammation  of  the  intestines),  and  sometimes  an  acute  serous 
pneumonia  is  also  present.  According  to  Ward  the  most  striking 
intestinal  lesions  are  found  deeply  in  the  first  and  second  duodenal 
flexures.  The  mucosa  is  deeply  reddened  and  studded  with  hemor- 
rhages, varying  in  size,  but  seldom  exceeding  one  millimeter  in 
diameter.  These  hemorrhages  involve  the  intestinal  coats  to  such  an 
extent  that  they  are  distinctly  visible  on  the  peritoneal  surface.  The 
contents  of  the  duodenum  consist  of  a  pasty  mass  more  or  less 
thickly  intermingled  with  blood  clots.  Marked  lesions  are  very  rarely 
observed  in  other  portions  of  the  intestines.  In  cases  where  these 
morbid  changes  are  not  so  well  marked,  diagnosis  with  the  naked 
eye  is  impossible,  and  the  blood  must  be  examined  microscopically 
and  other  birds  inoculated. 

Morphology  and  Staining  Properties. — The  organism  causing  fowl 
cholera  is  now  generally  known  as  the  Bacillus  avisepticus  (Latin, 
avisepticus,  septic  for  birds).  It  is  one  of  the  smallest  of  the  group  and 
is  rarely  longer  than  one  micron.  The  bacilli  are  frequently  seen  in 
the  blood  as  round  or  oval  bodies;  they  stain  with  the  ordinary  watery 
anilin  stains  in  a  polar  manner,  and  can  be  easily  mistaken  for  diplo- 
cocci.  They  are  Gram  negative,  immobile,  possess  no  flagella,  and 
form  no  spores.  Enormous  numbers  are  present  in  the  blood  of  sick 
and  dead  birds.  The  diagnosis  from  the  blood  must  be  made  either 
before  or  shortly  after  death,  because  putrefaction  bacteria  closely 
resembling  the  Bacillus  avisepticus  frequently  develop  in  cadavers  of 
birds. 

Cultural  Properties. — In  a  gelatin  stick  culture  made  from  the  blood  of 
a  bird  and  kept  at  room  temperature,  densely  crowded,  small,  translu- 
cent whitish  points  appear  after  a  few  days  along  the  line  in  the  gelatin. 
These,  later,  become  confluent  and  form  a  white  filiform  mass.  On 
the  surface,  small,  delicate,  transparent  dewdrop-like  colonies  appear, 
and  later  become  more  decidedly  white.  The  gelatin  does  not  become 
liquefied.  On  agar  slants  the  development  is  similar  but  quite  scanty, 
and  the  colonies  generally  do  not  become  confluent  but  remain  small. 
The  growth  on  blood  serum  is  generally  more  abundant,  and  leads  to 
a  thin,  dull  white  film  over  the  entire  surface.  The  organism  generally 
does  not  grow  on  potatoes.  The  growth  in  nutrient  bouillon  is  abun- 
dant. It  produces  clouding  of  the  medium,  a  sediment  at  the  bottom 
of  the  tube,  and  sometimes  a  thin,  delicate  pellicle  on  the  surface. 

Susceptible  Animals. — If  chickens,  geese,  ducks,  pigeons,  turkeys, 
sparrows,  etc.,  are  inoculated  subcutaneously  with  a  minimum  amount 
from  cultures  of  the  Bacillus  avisepticus,  they  develop  a  rapidly  fatal 
hemorrhagic  septicemia.  Rabbits  and  mice  are  also  quite  susceptible, 
and  succumb  to  the  infection.  Cattle,  sheep,  and  horses,  after  sub- 
cutaneous infection,  develop  local  abscesses  containing  numerous 
bacteria,  but  there  is  no  general  infection.  Guinea-pigs,  after  sub- 
cutaneous inoculation,  develop  a  local  abscess  only,  but  after  an 
intraperitoneal  application  die  from  a  septic  peritonitis.  Repeated 


BACILLUS  AVISEPTICUS  223 

passage  of  the  bacteria  through  the  bodies  of  susceptible  animals 
increases  its  virulency,  and  exposure  to  air  and  light  lessens  it. 

Natural  Infection.— Among  fowl,  infection  is,  as  a  rule,  transmitted 
through  the  gastro-intestinal  tract.  It  has  been  ascertained  that 
fowls  contract  the  disease  from  eating  infected  organs  and  food  soiled 
with  feces  from  infected  birds.  The  bacteria,  after  ingestion,  invade 
first  the  lacteals,  then  the  lymph  clefts,  and  from  them  the  blood 
current.  It  is  also  highly  probable  that  ectogenous  parasites  (lice) 
can  spread  the  disease  from  sick  to  healthy  birds. 

Resistance. — In  moist  soil  protected  against  air  and  light  the  Bacil- 
lus -avisepticus  can  remain  alive  and  virulent  for  a  considerable  time; 
in  manure,  according  to  Gartner,  at  least  one  month;  in  putrefying 
cadavers,  according  to  Kitt,  three  months.  It  is  not  killed  by  freezing, 
but  it  soon  perishes  when  dried  out.  It  loses  its  virulency,  according 
to  Kitt,  when  exposed  in  the  moist  condition  for  one-half  hour  to 
45°  to  46°  C.;  it  is  killed  at  from  80°  to  90°  C.  in  five  to  ten  minutes. 
Solomon  and  others  have  studied  the  effects  of  disinfectants  upon  the 
bacillus,  and  have  found  that  chlorinated  lime  in  a  dilution  of  1  to 
100;  slacked  lime,  1  to  20;  sulphuric  acid,  1  to  300;  hydrochloric 
acid,  1  to  500,  and  1  per  cent,  carbolic  acid  rapidly  kill  the  organism. 
Repeated  whitewashing  of  infected  places  is  an  excellent  means  of 
disinfection,  but  before  application  the  woodwork  should  be  washed 
with  hot  soda  solution  and  the  floors  scrubbed  with  creolin  or  lysol 
solution.  Sick  animals  must  be  separated  from  the  healthy,  and 
cadavers  should  be  deeply  buried,  or,  better  still,  burned.  According 
to  the  observation  of  several  investigators  the  bacilli  are  frequently 
found  as  saprophytes  in  the  outside  world. 

Immunization. — The  first  experiments  in  protective  inoculation 
against  this  bacillus  by  attenuated  cultures  were  made  by  Pasteur. 
Attenuated  cultures  powerful  enough  to  kill  pigeons  will  only  cause 
local  necrotic  processes  in  chickens,  geese,  and  ducks.  Passive  immu- 
nization has  been  practised  (Kitt)  with  a  serum  from  horses  hyper- 
immunized  against  the  Bacillus  avisepticus.  Jensen  has  observed 
that  chickens  which  have  passed  through  an  infection  with  the  bacillus 
of  the  hemorrhagic  septicemia  of  calves  were  subsequently  immune 
against  infection  with  the  Bacillus  avisepticus.  The  latter,  it  has 
been  claimed,  produces  a  soluble  toxin  which  will  pass  a  Pasteur  filter. 
Since  it  is  known,  however,  that  very"  small  bacilli  sometimes  pass 
through  a  Berkefeld  filter,  it  is  possible  that  the  transitory  effects  of 
the  filtrate  depended  upon  the  presence  of  a  very  few  not  very  virulent 
bacilli. 

Other  Septicemias  among  Birds. — In  addition  to  the  common  fowl 
cholera  a  number  of  other  septicemias  in  birds  have  been  described. 
They  are  evidently  not  identical  with  the  avisepticus  infection,  and 
are  caused  by  different  organisms.  Some  of  these  affections  are: 

A  disease  observed  among  pigeons  in  New  Jersey  by  Moore;  one 
observed  among  chickens  by  Noergaard  and  Mohler,  caused  by  a 


224     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

streptococcus  also  pathogenic  to  rabbits,  mice,  pigeons,  and  dogs;  and 
another  among  domestic  birds  caused  by  the  spirillum  of  Metchnikoff 
(see  under  spirilla).  Another  contagious  disease  of  chickens  with 
the  characteristics  of  a  septicemia  has  been  observed  in  Lombardy, 
Austria,  and  Germany.  It  is  transferable  through  the  blood  of  sick 
animals,  and  is  due  to  an  invisible,  filterable,  contagious  virus. 


BACILLUS   BOVISEPTICUS   (BACILLUS   OF   HEMORRHAGIC 
SEPTICEMIA  OF  BOVINES). 

Occurrence. — Bollinger,  in  1878,  first  described  a  disease  occurring 
in  Bavaria  among  wild  deer  and  wild  hogs,  which  later  spread  to 

FIG.  125 


Interior  of  right  ventricle  of  the  heart  of  a  cow  showing  hemorrhagic  endocarditis  as  it  occurs 
in  hemorrhagic  septicemia.     (Reynolds.) 


domestic  cattle  and  hogs,  and  even  horses.    While  he  recognized  its 
infectious  character,  Kitt,  in  1885,  was  the  first  to  discover  its  cause, 


BACILLUS  BOVISEPTICUS  225 

and  Hiippe,  in  1886,  to  study  it  in  detail.  The  disease  has  since  been 
found  in  various  parts  of  Germany,  Austria,  and  other  countries 
of  Europe,  the  United  States,  Indo-China,  the  Malayan  Peninsula, 
Java,  Hongkong,  the  Philippine  Islands,  and  also  in  Algiers,  in  Africa. 

Pathologic  Lesions. — The  pathologic  lesions  are  those  of  a  hemor- 
rhagic  septicemia;  namely,  general  congestion,  petechial  hemorrhages, 
and  ecchymoses  into  the  mucous  and  serous  membranes  and  internal 
organs.  The  liver  and  kidneys  are  swollen  and  cloudy,  but  the  spleen, 
unlike  its  condition  in  anthrax  with  which  this  hemorrhagic  septi- 
cemia may  be  confounded,  is  not  enlarged,  and  has  either  a  general 
normal  appearance  or  contains  multiple,  circumscribed  hemorrhages. 
In  the  so-called  exanthematous  or  edematous  form  the  subcutaneous 
connective  tissue  of  the  head  and  neck  contains  a  watery  and  partly 
hemorrhagic  infiltration.  The  tongue  is  frequently  more  or  less 
enlarged,  dark  or  dirty  brown  red,  and  of  a  firm  consistency.  The 
mucous  membranes  of  the  mouth  and  upper  respiratory  tract  and 
the  retropharyngeal  and  cervical  glands  are  swollen.  The  mucosa  of 
the  intestinal  tract  is  also  swollen  and  hemorrhagic.  In  some 
cases  the  lungs  show  not  merely  congestion,  but  areas  of  consoli- 
dation, and  the  pericardium,  in  addition  to  the  pericardial  hemor- 
rhages, may  show  the  signs  of  a  fibrinous  inflammation.  Reynolds 
reported  an  outbreak  of  the  disease  in  Minnesota  in  which  meningeal 
lesions  of  the  brain  and  cord  were  very  marked.  The  disease  is  caused 
by  the  Bacillus  pluriformis  of  Kitt,  now  more  generally  known  as  the 
Bacillus  bovisepticus. 

Morphology  and  Staining  Properties. — The  bacillus  possesses  the 
same  morphologic  and  staining  properties  as  the  Bacillus  avisep- 
ticus.  It  is  from  0.6  to  1  micron  long,  sometimes  a  little  larger,  and 
0.3  micron  wide.  It  is  found  in  considerable  numbers  in  the  blood 
of  animals  in  the  last  stages  of  the  disease  or  in  those  dead  from  it, 
and  in  enormous  numbers  in  the  blood  of  artificially  infected  rabbits 
or  mice,  which  are  exceedingly  susceptible.  It  often  appears  in  the 
blood  in  short  chains.  In  doubtful  cases  the  diagnosis  should  not 
be  made  until  rabbits  or  mice  have  been  inoculated.  These  die  in 
from  twelve  to  twenty-four  hours. 

Cultural  Properties. — Wilson  and  Brimhall  describe  the  cultural 
properties  as  follows :  The  organism  is  aerobic,  but  prefers  the  depth 
rather  than  the  surface  of  the  medium.  It  grows  best  at  the  body 
temperature  and  more  slowly  at  room  temperature.  In  ordinary 
nutrient  and  in  glucose  bouillon  a  heavy  growth  appears  in  twenty- 
four  hours.  In  Dunham's  solution  a  small  amount  of  indol  is  formed 
in  forty-eight  hours.  Milk  is  not  coagulated.  In  gelatin  plates  small, 
white,  granular  colonies  appear  after  forty-eight  hours.  In  gelatin 
stab  cultures  a  light  growth  occurs  on  the  surface,  while  along  the 
needle  track  numerous  colonies  like  those  in  the  deep  portions  of  the 
plate  develop  in  the  culture.  The  organism  grows  on  neutral  but 
not  on  acid  potatoes. 
15 


226      BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

Resistance. — The  organisms  are  destroyed  in  fluids  at  58°  C.  in 
seven  or  eight  minutes;  by  corrosive  sublimate  solution  1  to  5000  in 
one  minute. 

Natural  Infection. — The  common  mode  in  cattle  seems  to  be  through 
the  intestinal  tract.  It  appears  that  the  Bacillus  bovisepticus  is  wide- 
spread as  a  saprophyte  in  nature,  and  that  it  may  lose  most  of  its 
virulency,  but  also  regain  it  under  conditions  which  are  not  yet  well 
known. 

Other  Septicemias  among  Bovines. — Corn-fodder  Disease. — It  was 
formerly  believed  that  the  so-called  corn-fodder  or  corn-stalk  disease, 
which  sometimes  occurs  extensively  among  cattle  along  the  upper  and 
middle  Mississippi  Valley,  was  due  to  the  Bacillus  bovisepticus,  but 
according  to  Moore's  investigations  this  certainly  does  not  appear  to 
be  the  case. 

Septic  Pleuropneumonia  of  Calves. — This  disease  probably  differs 
only  clinically  from  the  typical  hemorrhagic  septicemia  of  cattle,  and 
is  due  to  the  Bacillus  bovisepticus,  though  it  has  been  claimed  that 
it  is  caused  by  a  somewhat  different  variety. 

Hemorrhagic  Septicemia  of  Sheep. — This  disease  has  been  observed 
in  Europe  and  Argentina.  It  presents  the  typical  pathologic  changes 
seen  in  the  other  hemorrhagic  septicemias  due  to  the  bacilli  of  this 
group.  It  is  caused  by  the  variety  known  as  Bacillus  (bipolaris) 
ovisepticus. 

BACILLUS  SUISEPTICUS. 

Hemorrhagic  septicemia  of  swine,  caused  by  the  Bacillus  suisep- 
ticus,  is  commonly  known  as  swine  plague.  There  was  considerable 
confusion  in  the  past  as  to  the  exact  etiology  and  pathology  of  this 
disease,  because  it  is  frequently  associated  with  another  disease  of 
swine  known  as  hog  cholera,  and,  further,  because  both  diseases  may 
occur  as  a  mixed  infection  in  a  single  animal. 

The  nomenclature  is  another  unfortunate  and  confusing  feature. 
The  English  word  "swine  plague"  literally  translated  into  German  is 
"Schweinepest,"  just  as  the  English  term  bubonic  plague  is  "Beulen- 
pest."  The  German  word  "Schweinepest,"  however,  is  the  name  for 
the  disease  called  hog  cholera  in  English  and  not  for  swine  plague. 
This  explains  why  so  much  confusion  has  arisen  in  the  discussion  of 
these  two  diseases  of  swine.  Swine  plague  is  due  to  a  bacillus  of  the 
hemorrhagic  septicemia  group,  while  hog  cholera,  formerly  believed 
to  be  due  to  a  bacillus  of  the  typhoid  colon  group  (the  hog  cholera 
bacillus),  is  most  probably  due  to  an  invisible,  filterable  virus., 

Historical  and  Occurrence. — Loeffler,  in  1886,  discovered  that  the 
septicemic  form  of  swine  plague  was  due  to  a  specific  organism  which 
was  later  classified  with  the  other  bacilli  of  the  bipolar  group.  Schiitz, 
in  the  same  year,  demonstrated  the  presence  of  the  identical  bacillus 
in  the  pectoral  form  of  the  affection.  The  later  contributions  by 


BACILLUS  SUISEPTICVS  227 

Salmon,  Smith,  Moore,  Jensen,  and  Bang  confirmed  the  early  work 
of  Loeffler  and  Schiitz.  The  disease  has  been  found  in  several  Euro- 
pean countries,  and  is  quite  widely  spread  throughout  the  United 
States. 

Pathologic  Lesions. — In  the  most  acute  cases  the  changes  found  in 
the  dead  animals  are  those  of  a  typical  hemorrhagic  septicemia,  with 
petechial  and  ecchymotic  hemorrhages  into  the  skin,  subcutaneous 
fat,  and  the  serous  and  mucous  membranes.  The  kidneys,  including 
the  subscapular  tissue,  the  pelves,  and  even  the  parenchyma,  very 
frequently  are  the  seat  of  blood  extravasation.  The  membranes  of 
the  brain  are  likewise  frequently  hemorrhagic;  the  lymph  glands 
show  a  condition  of  acute  hemorrhagic  inflammation.  In  cases  which 
have  not  taken  the  most  acute  course  the  lungs  are  generally  found 
involved.  They  show  multiple  areas  of  consolidation  which  are  at 
first  dark  brown  red  and  later  a  lighter  grayish  red.  The  foci  of 
consolidation  contain  necrotic  material,  and  in  older  cases  the  entire 
lobe  of  a  lung  may  have  become  changed  into  a  mass  of  caseous 
necrotic  substance.  The  non-consolidated  pulmonary  tissue  is  edem- 
atous,  the  pleura  thickened,  congested,  and  covered  by  a  fibrinous 
exudate.  The  pericardium  may  show  similar  changes,  and  the  pleural 
cavity  may  contain  a  varying  amount  of  hemorrhagic  seropurulent 
fluid.  The  mucosa  of  the  gastro-intestinal  tract  is  swollen  and  hemor- 
rhagic and  sometimes  covered  by  rather  thin pseudomembranes,  formed 
by  superficial  necrotic  cells.  The  spleen  is  not  enlarged,  the  kidneys 
are  congested.  According  to  Moore  the  pneumonia  of  swine  plague 
presents  itself  both  as  a  lobar  and  as  a  lobular  bronchopneumonia. 
In  the  former  the  alveoli  become  filled  with  red  and  white  blood  cor- 
puscles and  fibrin.  In  chronic  cases  the  animals  are  much  emaciated, 
and  the  lungs,  peribronchial  and  mesenteric  glands  and  tonsils  contain 
necrotic  foci. 

Morphology  and  Staining  Properties. — In  the  most  acute  cases  the 
Bacillus  suisepticus  (also  called  Bacterium  suicidum)  is  found  in 
great  numbers  in  the  blood  and  internal  organs.  In  the  less  acute 
cases  it  is  found  in  the  consolidated  and  necrotic  foci  in  the  lungs, 
occasionally  also  in  the  blood.  In  the  chronic  cases  the  organisms 
do  not  occur  in  the  blood,  but  as  necrotic  foci  in  combination  with 
other  bacteria,  such  as  streptococci,  staphylococci,  Bacillus  pyogenes 
suis,  Bacillus  necrophorus,  and  others.  The  bacillus  suisepticus  is 
one  micron  to  a  micron  and  a  fraction  long,  0.5  to  0.6  wide.  It  is 
sometimes  almost  oval  or  round;  at  times  it  stains  quite  uniformly, 
at  other  times  in  a  bipolar  manner.  Longer  bacilli  occasionally  stain 
like  the  diphtheria  bacillus,  at  both  ends  and  in  the  centre.  The 
bacillus  stains  with  the  ordinary  anilin  stains,  is  Gram  negative,  not 
motile,  possesses  no  flagella,  but  a  capsule  can  be  demonstrated  by 
proper  staining  methods.  The  organism,  like  that  of  bubonic  plague 
in  older  lesions,  is  often  of  a  swollen  vacuolated  type,  stains  only  with 
a  peripheral  ring,  and  then  has  the  appearance  of  an  empty  shell. 


228     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

Bang,  therefore,  called  it  the  vacuole  bacillus.  Loeffler  has  described 
a  Bacillus  parvus  ovalus  which  was  probably  a  Bacillus  suisepticus  of 
this  vacuolated  type. 

Cultural  Properties. — The  Bacillus  suisepticus  grows  at  room  tem- 
perature in  artificial  culture  media,  particularly  gelatin.  It  grows  well 
on  coagulated  blood  serum  in  the  incubator,  not  so  well  on  agar  or 
in  bouillon.  When  the  growth  on  agar  is  more  abundant  it  forms  a 
sticky,  slimy  layer.  It  generally  does  not  develop  on  potatoes.  It 
sometimes  forms  indol  and  sometimes  phenol. 

Animals  Susceptible. — Mice  and  rabbits  are  very  susceptible.  They 
can  be  easily  infected  from  a  small  wound,  and  die  very  rapidly 
after  the  inoculation.  Guinea-pigs,  pigeons,  and  chickens  sometimes 
succumb  to  the  infection,  at  other  times  they  are  not  susceptible. 

Resistance. — According  to  Moore  and  Smith  the  resistance  of  the 
Bacillus  suisepticus  to  physical  and  chemical  agencies  is  as  follows: 
It  is  killed  if  heated  in  bouillon  for  ten  minutes  to  58°  C.  Complete 
drying  out  destroys  it  in  twenty-four  to  thirty-six  hours.  It  does 
not  live  in  soil  over  a  week  and  in  water  not  over  eleven  days.  It  is 
killed  in  lime  water  in  one  minute,  in  1  per  cent,  carbolic  acid  in  five 
minutes,  and  in  formalin,  1  to  1000,  in  five  minutes.  The  organism 
is,  accordingly,  not  very  resistant. 

Immunization. — The  first  experiments  in  the  immunization  of  small 
laboratory  animals  and  swine  against  the  Bacillus  suisepticus  by 
attenuated  cultures  were  made  by  de  Schweinitz  and  Smith.  The 
latter,  in  1891-92,  reported  successful  immunization  of  rabbits  and 
swine,  and,  in  association  with  Moore,  again  in  1894.  Ostertag  and 
Wassermann  have  shown,  independently,  that  animals  immunized 
with  a  certain  stem  of  Bacillus  suisepticus  are  subsequently  immune 
only  against  this  stem,  and  not  against  any  other  stem.  At  best  they 
are  only  immune  against  a  few  stems,  and  never  against  all  that 
might  subsequently  be  employed.  This  condition,  which  was  con- 
firmed by  others,  is  not  absolutely  without  a  parallel,  as  it  is  met 
with  also  in  certain  bacilli  of  the  coli  group.  Ostertag  and  Wasser- 
mann later  prepared  a  polyvalent  antiswine  plague  serum  by  inject- 
ing a  number  of  horses,  each  with  several  stems,  and  then  mixing  the 
sera  of  the  different  horses.  According  to  these  authors,  and  also 
Joest,  Raebinger,  and  others,  the  use  of  the  polyvalent  serum  has  been 
very  satisfactory  in  Germany.  From  other  sources  the  reports  have 
not  been  so  favorable. 


BACILLUS  EQUISEPTICUS. 

Equine  influenza,  known  also  under  the  names  of  pink-eye,  typhoid 
fever,  epizootic  catarrhal  fever,  horse  distemper,  mountain  fever, 
"Pferdestaube,"  "Rothlaufseuche,"  and  "Brustseuche"  (German); 
Pasteurelosis  equorum,  la  grippe,  fievre  typhoid,  pneumonia 


BACILLUS  EQUISEPTICUS  229 

infectieuse  (French),  is  a  disease  of  horses  claimed  by  a  number  of 
observers  to  bs  due  to  the  Bacillus  (bipolaris)  equisepticus. 

Occurrence. — The  disease  occurs  sporadically,  and  also  as  wide- 
spread epidemics.  It  has  been  observed  in  Europe,  America,  and 
South  Africa.  It  became  prevalent  in  the  eastern  part  of  the  United 
States  in  1872-3,  and  from  there  has  spread  over  the  entire  country. 

Pathologic  Lesions. — In  the  catarrhal  form  the  mucous  membranes 
of  the  respiratory  and  gastro-intestinal  tract  show  an  intense  con- 
gestion often  accompanied  by  edematous  infiltrations,  particularly 
in  the  submucous  connective  tissue  of  the  larynx,  the  pylorus,  and  the 
small  intestines.  The  subcutaneous  connective  tissues  are  likewise 
often  infiltrated  with  a  watery  exudate.  Peyer's  patches  of  the  small 
intestine  and  the  mesenteric  glands  are  swollen,  and  are  reddish  gray 
on  section.  The  pleural  and  peritoneal  cavities  frequently  contain  a 
reddish,  cloudy,  serous  fluid.  The  spleen  shows  some  swelling,  and 
the  lungs  are  edematous.  In  the  pectoral  form  the  lungs  contain  lobar 
or  lobular  areas  of  consolidation,  and  the  pleura  is  in  a  condition  of 
serofibrinous  inflammation.  The  consolidated  areas  in  the  lungs 
become  necrotic,  and  contain  a  soft,  dirty  grayish-brown  or  greenish, 
very  fetid  mass.  The  necrotic  foci  sometimes  break  through  the 
pleura  and  establish  a  pneumothorax  or  pyopneumothorax.  In  the 
catarrhal  form  the  conjunctive  of  the  eyes  are  intensely  reddened1 
(brick  red  or  mahogany  red),  and  the  submucous  connective  tissue 
is  edematous.  The  eyelids  swell,  covering  the  eyeball  to  a  large 
extent.  If  the  lids  are  opened  forcibly  there  is  an  abundant  flow  of 
tears,  and  later  a  mucopurulent  discharge. 

Morphology. — The  Bacillus  equisepticus  is  described  by  Nocard 
and  Leclainche,  according  to  Ligniere's  findings,  as  follows:  It  is  a 
slender,  short  bacillus  with  rounded  ends  and  of  about  the  same  size 
and  staining  properties  as  the  bacillus  of  fowl  cholera.  In  cultures  it 
shows  as  a  very  small  diplococcus  (coccobacillus).  It  is  immobile, 
strictly  aerobic,  and  Gram  negative.  It  is  present  in  the  blood  in 
small  numbers  only,  and  difficult  to  find. 

Cultural  Properties. — Artificial  cultures  are  difficult  to  obtain.  They 
require  the  intraperitoneal  injection  of  4  or  5  c.c.  of  the  horse's 
blood,  pleuritic  fluid  or  juice  from  the  lungs  into  a  guinea-pig,  in 
which  they  produce  a  peritonitis,  the  exudate  from  which  contains 
numerous  ovoid  bacteria,  from  which  culture  media  are  inoculated. 
Bouillon  cultures,  after  twenty-four  hours,  are  uniformly  clouded,  and 
the  reaction  of  the  medium  remains  the  same  even  after  several  days. 
On  gelatin  at  20°  C.  very  small,  round,  almost  transparent  colonies 
are  formed  after  two  or  three  days.  The  bacillus  does  not  grow  well 
on  plain  or  glycerin  agar;  it  grows  in  milk  without  coagulating  it; 
no  growth  occurs  on  potatoes.  The  best  culture  medium  is  bouillon 
to  which  a  small  amount  of  sterile  horse's  blood  serum  has  been  added. 

1  The  name  pink-eye  is  derived  from  this  symptom. 


230     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

The  organism  is  pathogenic  for  guinea-pigs,  rabbits,  mice,  dogs, 
cats,  swine,  sheep,  donkeys,  and  horses.  Whether  it  is  pathogenic 
for  cattle  is  doubtful. 

Natural  Infection. — In  the  horse,  natural  infection  is  spread  by  the 
secretion  from  the  diseased  mucous  membranes  and  lungs  and  through 
the  feces.  These  secretions  and  excretions  are  particularly  infectious 
while  the  disease  is  at  its  height,  but  they  may  also  spread  the  con- 
tagium  for  a  long  time  after  the  affection  has  run  its  course.  After 
infection  of  a  previously  healthy  animal  the  bacilli  multiply  rapidly 
in  the  mucous  membranes  and  invade  the  lymph  and  blood  circula- 
tions, producing  in  very  violent  rapidly  fatal  cases  the  picture  of 
typical  hemorrhagic  septicemia. 

Hutyra  has  confirmed  the  observations  of  Ligni£re,  identifying  the 
Bacillus  equisepticus  as  the  cause  of  horse  influenza,  or  pink-eye,  but 
other  authors  still  consider  the  etiology  unsettled,  and  doubt  whether 
this  organism  is  the  actual  cause. 


BACILLUS  OF  DOG  TYPHUS. 

An  acute,  epidemic  disease  of  dogs,  characterized  by  a  violent 
gastro-enteritis  with  ulcerative  stomatitis  was  first  described  in  1850 
by  Hofer  under  the  name  of  "Hundetyphus,"  dog  typhus,  and  later 
under  the  name  gastro-enteritis  hemorrhagica.  During  the  last  two 
decades  several  larger  epidemics  have  been  described  in  Germany 
and  other  parts  of  Europe.  Ligniere  claims  that  the  disease  is  due 
to  a  bacillus  of  the  hemorrhagic  septicemia  group.  He  describes  the 
organisms  as  having  the  general  characteristics  of  the  group,  but  that 
it  is  larger  when  it  first  occurs  in  the  dog  and  only  becomes  smaller 
after  having  passed  through  several  guinea-pigs.  It  grows  on  culture 
media  much  like  the  other  members  of  the  group.  Ligniere's  claim 
has  not  been  confirmed.  Hutyra  states  that  he  has  examined  bacte- 
riologically  a  number  of  cases,  but  has  not  obtained  Ligniere's  bacillus 
but  a  bacillus  of  the  colon  group  and  another  virulent  bacillus  of  the 
proteus  group. 

BACILLUS  PESTIS. 

Occurrence  and  Historical. — The  Bacillus  pestis,  or  the  bacillus  of 
bubonic  plague,  is  the  most  extensively  studied,  and  now  best-known 
organism  of  the  group  of  bacilli  of  hemorrhagic  septicemias.  It  is  the 
cause  of  the  most  dreaded  human  scourge,  bubonic  plague,  the  "Great 
Black  Death"  of  the  Middle  Ages,  which  is  estimated  to  have  carried 
away,  within  a  space  of  three  years,  twenty-five  millions  of  people  in 
Europe  during  the  fourteenth  century.  In  the  last  century  the  disease 
seemed  to  have  almost  if  not  completely  died  out,  but  toward  the  close 
a  great  pandemic  broke  out  in  India  and  China.  It  spread  to  various 


BACILLUS  PESTIS  231 

places  in  Asia,  including  the  Philippine  Islands,  to  Europe,  Africa, 
Australia,  and  also  invaded  the  western  shores  of  the  United  States. 
The  great  epidemic  in  India  and  China  still  exists,  and  is  annually 
killing  hundreds  of  thousands  of  people.  The  disease  is  particularly 
interesting  to  the  veterinarian,  not  merely  from  a  general  human,  but 
from  a  special  professional  standpoint,  because  it  is  a  natural  disease 
of  some  rodents,  more  especially  of  the  various  varieties  of  the  common 
rat.  That  rats  die  extensively  during  plague  epidemics  had  been 
noticed  in  the  Middle  Ages,  and  certain  statements  occur  in  the  Bible 
which  evidently  refer  to  rodents  dying  from  plague.  The  investi- 
gations in  particular  of  the  English  Indian  Plague  Commission  and 
the  Advisory  Committee  for  Plague  Investigation  in  India  have 
demonstrated  the  great  prevalence  of  acute  plague  among  the  rats 
and  its  relation  to  the  spread  of  the  disease  to  man. 

Pathologic  Lesions  of  Plague  in  Rats. — It  is  of  the  greatest  practical 
importance  to  recognize  plague  in  rats.  It  frequently,  if  not  generally, 
precedes  plague  in  man,  and  the  human  disease  cannot  be  stamped 
out  successfully  in  a  territory  unless  it  is  eliminated  among  the  rats. 
Fortunately,  plague  in  rats  can  be  diagnosticated  easily  and  generally 
by  means  of  the  naked  eye  without  an  additional  microscopic  examina- 
tion. The  plague  bacillus,  both  in  rats,  other  animals,  and  man,  tends 
to  invade  the  lymph  glands  nearest  to  its  place  of  entrance,  and  there 
multiplies  enormously,  leading  to  very  characteristic  changes,  such  as 
swelling,  edema,  and  hemorrhages  into  the  gland  and  the  neighbor- 
hood of  the  periglandular  region.  Later  the  gland  may  contain  pus 
or  a  necrotic  caseous  material.  A  gland  presenting  the  indicated 
pathologic  changes  is  called  a  bubo,  and,  hence,  the  disease,  which  so 
regularly  leads  to  the  formation  of  such  buboes,  is  known  as  bubonic 
plague.  The  gland  lying  closest  to  the  place  of  infection  and  first 
suffering  marked  pathologic  changes  is  called  the  primary  bubo  of  the 
first  order.  If  neighboring  glands  are  affected  by  direct  continuous 
transport  of  the  bacilli  they  are  known  as  primary  buboes  of  the  second 
order.  The  distant  glands  which  become  infected  through  the  blood 
circulation  or  through  the  lymph  circulation  are  known  as  secondary, 
tertiary  buboes,  etc.  The  Advisory  Committee  for  Plague  Investiga- 
tion reported  on  the  examination  of  over  31,000  rats  in  Bombay, 
India,  4000  as  infected  with  plague.  The  postmortem  findings  fur- 
nished the  following  general  picture.  Subcutaneous  congestion,  visible 
after  removing  the  skin  of  the  animal,  is  not  infrequently  a  well- 
marked  feature.  It  may  be  general,  but  in  some  cases  is  limited  to 
the  bubo.  A  peculiar  purplish-red  appearance  of  the  muscles  exposed 
by  reflecting  the  skin  of  the  thorax  and  abdomen  is  obviously  due  to 
the  presence  of  congested  vessels,  and  combined  with  the  reddish- 
pink  color  of  the  subcutaneous  tissue  is  a  strong  indication  of  plague 
at  the  beginning  of  the  examination.  Subcutaneous  hemorrhages  are 
frequently  noticed  particularly  in  the  submaxillary  region;  here  also 
edema  is  common;  general  edema  over  the  entire  body  is  quite  rare. 


232      BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

In  a  healthy  rat  the  only  glands  which  are  large  enough  to  be  seen 
easily  are  those  forming  the  crescent  embracing  the  salivary  glands 
in  the  submaxillary  region,  and  the  elongated  retroperitoneal  glands 
on  each  side  of  the  middle  line  in  the  lower  part  of  the  abdomen. 
The  latter  are  called  the  pelvic  glands.  In  the  bubonic  type  of  plague 
the  glands  affected  and  presenting  the  picture  of  the  typical  plague 
bubo  described  above  are  in  the  order  of  frequency  of  infection, 
according  to  the  Bombay  figures :  Glands  of  the  neck  (75  per  cent.), 
glands  of  the  axilla  (15.1  per  cent.),  glands  of  the  groin  (6.1  per  cent.), 
glands*of  the  pelvis  (3.8  per  cent.).  A  bubo  in  a  plague-infected  rat 
feels  hard  when  cut  across,  but  it  has  not  the  tough  consistency  of  a 
normal  gland.  The  contents  of  the  latter  are  not  easily  squeezed  out 
by  pressure,  while  in  a  bubo  the  substance  of  the  gland  is  readily 
broken  down  by  slight  pressure  with  the  forceps.  A  bubo  on  section 
has  the  appearance  of  necrosis,  affecting  first  the  medullary  portion 
of  the  gland  and  gradually  spreading  outward,  so  that  ultimately  the 
gland  may  be  converted  into  a  mass  of  necrotic  tissue  enclosed 
within  the  capsule.  The  central  portion  consequently  appears  gray, 
and  at  a  later  stage  the  centre  breaks  down  into  a  rather  dry,  very 
rarely  a  liquid,  purulent  material.  Buboes  with  greenish  liquid  pus 
are  not  typical  of  plague. 

The  presence  of  a  typical  bubo  is  the  most  important  sign  of  plague 
in  rats,  and  next  in  importance  is  the  so-called  granular  liver.  Accord- 
ing to  the  Bombay  findings,  it  is  only  met  with  in  rats  infected  with 
plague.  The  liver  appears  to  be  in  a  condition  of  fatty  degeneration, 
but,  as  a  matter  of  fact,  the  changes  are  not  fatty  but  necrotic.  The 
outlines  of  the  lobules  are  distinct  and  the  surface  of  the  organ  presents 
numerous  gray  or  whitish  granules  of  the  size  of  a  pinpoint,  which 
give  the  liver  a  stippled  appearance  as  if  it  had  been  dusted  over  with 
gray  pepper.  This  appearance  has  given  rise  to  the  term  granular 
liver.  The  gray  areas  may  be  so  small  that  only  the  closest  scrutiny 
of  an  experienced  observer  will  detect  them.  When  larger  the  granules 
are  of  a  yellow  color  and  vary  somewhat  in  size.  In  a  typical  case 
the  granules  are  not  raised  above  the  surface  of  the  liver.  The  spleen, 
which  is  very  typically  changed  in  experimental  plague  in  guinea-pigs, 
is  not  very  characteristically  changed  in  rat  plague,  though  it  is  gener- 
ally markedly  enlarged.  One  of  the  most  important  postmortem 
findings  in  the  disease  is  the  presence  of  an  abundant  clear  pleural 
effusion.  Hemorrhages,  both  subcutaneous,  subperitoneal,  and  in 
the  internal  organs,  are  likewise  constant  and  important  pathologic 
changes  in  rat  plague. 

McCoy  has  recently  published  the  results  of  the  investigation  of 
rat  plague  in  California,  and  his  findings  on  comparatively  small 
material,  in  general,  agree  well  with  the  Bombay  findings.  However, 
in  the  American  material  the  bubo  was  generally  found  in  the  groin 
and  not  in  the  cervical  glands.  Since  the  quantity  of  material  exam- 
ined in  California  is  only  about  1  per  cent,  of  the  material  examined 


BACILLUS  PESTIS  233 

in  Bombay,  the  figures  from  the  former  have  not  much  influence  on 
the  figures  from  India.  McCoy  has  once  or  twice  seen  rats  dead  from 
plague  which  did  not  show  any  of  the  characteristic  pathologic 
changes.  In  these  cases  the  presence  of  the  plague  bacillus  was 
established  by  animal  inoculation. 

Plague-infected  rats  generally  die  in  a  few  days,  but  sometimes, 
though  rarely,  the  plague  runs  a  chronic  course.  The  Indian  Plague 
Commission  encountered  a  considerable  number  of  these  chronic 
cases  in  two  villages  in  the  Punjab  Province. 

Animals  Susceptible. — According  to  Simpson,  Hunter,  and  others, 
rats  are  by  no  means  the  only  animals  susceptible  to  natural  plague 
infection;  these  authors  also  mention  pigs,  calves,  sheep,  monkeys, 
geese,  ducks,  turkeys,  hens,  pigeons,  and  quails.  It  is,  however,  very 
probable  that  this  claim  is  not  quite  correct,  and  that  other  members 
of  the  group  of  hemorrhagic  septicemia  bacilli  (Bacillus  bipolaris) 
have  been  mistaken  for  the  Bacillus  pestis.  Other  rodents  which, 
according  to  Blue,  have  been  shown  to  harbor  the  Bacillus  pestis,  and 
to  be  a  subject  to  plague  infection,  are  the  tarbegan,  a  species  of 
arctomynse,  found  in  Siberia;  the  marmot,  a  hibernating  rodent  of 
India  and  China;  the  marmot  of  Thibet,  the  tree  squirrel,  and  the 
California  ground  squirrel  (Otosperphilus  beecheyi).  According  to 
Blue  and  Wherry  four  plague-infected  ground  squirrels  were  found 
in  California  during  the  summer  of  1907,  and  it  was  possible  to 
show  that  several  persons  had  contracted  plague  directly  from  this 
animal. 

Spreading  of  the  Infection. — Plague  infection  in  man  and  animals  is 
generally  a  wound  infection,  near  which  most  cases  present  plague 
buboes.  A  certain  percentage  of  cases  are  probably  due  to  inhalation 
and  lead  to  the  pneumonic  type.  It  is  now  claimed,  particularly  upon 
the  basis  of  the  early  investigations  of  Simond  and  the  more  recent 
and  extensive  work  of  the  Indian  Plague  Commission  and  the  Advisory 
Plague  Committee  that  the  rat  flea  is  responsible  for  the  spread  of 
plague  from  rat  to  rat  and  from  rat  to  man.  This  flea  most  common 
on  rats  in  tropical  and  subtropical  countries  has  been  described  by 
Rothschild  as  Pulex  chceops.  About  the  same  time  the  author,  in 
ignorance  of  Rothschild's  work,  independently  found  and  described 
the  rat  flea  in  Manila  under  the  name  of  Pulex  philippinensis.  It 
is  now  claimed  that  the  rat  flea  will  frequently  bite  man,  although  in 
the  author's  experiments  it  did  not.  If  plague  is  conveyed  from  rat 
to  rat  by  the  flea,  it  is  certainly  strange  that  most  of  the  buboes  in  the 
rats  in  India,  i.  e.,  75  per  cent.,  are  found  in  the  cervical  region,  which 
would  indicate  that  fleas  bite  rats  most  commonly  on  the  head  and 
not  on  the  body.  The  very  frequent  occurrence  of  the  cervical  bubo 
was  formerly  explained  on  the  assumption  that  rats  generally  infected 
themselves  by  small  abrasions  or  wounds  in  the  mouth.  It  is  also 
well  known  that  even  among  well-fed  white  rats  the  living  eat  the 
dead  of  their  own  species. 


234     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

Plague  Bacilli  in  the  Blood. — It  appears  that  plague  in  rats  generally 
leads  to  an  early  true  septicemia,  that  is,  to  an  invasion  of  the  blood 
and  a  multiplication  of  the  infecting  bacilli.  The  Indian  investigators 
in  experimental  rat  inoculation  have  found  as  many  as  1,000,000,000 
bacilli  per  cubic  centimeter  of  blood.  This  exceptional  figure  was, 
however,  found  only  twice;  other  figures  were  as  low  as  10  to  100  per 
cubic  centimeter.  While  one  thousand  million  bacilli  appears  like 
an  excessively  high  figure,  it  is  not  really  so  in  view  of  the  fact  that 
one  cubic  centimeter  of  rat's  blood  contains  10,000,000,000  red  blood 
corpuscles,  and  that  in  some  of  the  other  hemorrhagic  septicemias, 
particularly  those  in  birds,  the  bipolar  bacilli  are  often  many  times 
in  excess  of  the  number  of  red  blood  corpuscles. 

FIG.  126 


Bacterial  embolism  in  a  vessel  of  the  kidney  (centre  of  field).    Human  infection  by  the  bacillus 
of  bubonic  plague.     (Author's  preparation.) 


Plague  in  Man. — According  to  the  author's  observations,  this  is 
generally  not  a  septicemia  but  a  local  infection  with  a  general  toxemia. 
If  blood  in  amounts  of  several  cubic  centimeters  is  obtained  from 
plague  patients  and  properly  incubated  in  fluid  culture  media  a  growth 
is  often  obtained,  but  this  may  be  due  to  the  presence  of  a  very  few 
bacilli,  and  it  only  proves  that  a  few  live  bacilli  were  in  the  blood  at 
the  time  it  was  taken.  In  a  true  septicemia,  however,  a  multiplication 
of  the  bacteria  in  the  blood  must  be  demonstrable.  True  hemorrhagic 
plague  septicemia  and  pyemia  with  the  formation  of  bacterial  emboli 


BACILLUS  PESTIS  235 

occurs  in  man  occasionally,  as  it  frequently  occurs  in  rats,  both 
naturally  and  experimentally. 

Latent  Plague. — It  was  Hunter,  of  Hongkong,  who  undertook  to 
explain  the  seasonal  appearance  of  plague  among  animals  and  man 
by  attempting  to  show  the  existence  of  a  latent  form  of  plague  in 
which  small  numbers  of  bacilli  were  present  in  the  blood,  but  for  the 
time  being  no  multiplication  nor  development  of  symptoms  of  disease. 
From  such  latent  cases  outbreaks  of  epidemics  might  originate. 
Such  conditions,  of  course,  prevail  in  Texas  fever  and  trypanosomiasis 
of  cattle.  Herzog  and  Hare,  investigating  this  question  in  the  Philip- 
pine Islands,  have  shown  that  latency  in  plague  does  not  occur. 

Morphology  and  Staining  Properties. — The  plague  bacillus  was  dis- 
covered simultaneously  and  independently  in  1894  by  Kitasato  and 
Yersin. 

It  is  relatively  variable  in  morphology,  a  fact  which  it  is  important 
to  remember  in  connection  with  the  bacteriologic  diagnosis  of  the 
disease.  In  postmortem  smears  prepared  from  a  recent  non-suppur- 
ating primary  bubo,  a  pneumonic  focus,  the  spleen,  and  occasionally 
from  other  internal  organs  of  both  man  and  rats,  numerous  plague 
bacilli  are  generally  found.  The  simplest  staining  method  for 
plague  bacilli  in  smears  and  in  pus,  necrotic  material,  etc.,  is  dilute 
carbol-fuchsin  (1  part  of  the  original  stain  to  5  to  10  aqua  destillata) 
for  twenty  to  forty  seconds  and  then  washing  freely  in  water. 

In  smears  made  from  the  organs,  the  plague  organism  appears  as 
a  rather  short,  plump  bacillus,  being  1.5  to  1.75  micra  long  and 
0.5  to  0.75  micron  thick;  generally  the  proportion  of  width  to  length 
is  as  one  to  two.  Individuals  considerably  longer  than  1.75  micra 
are  occasionally  seen.  The  bacilli  are  generally  single,  occasionally 
diplobacilli,  and  very  rarely  in  short  chains.  In  smears  which  have 
been  fixed  in  absolute  alcohol  and  properly  dyed  the  bacilli  are  not 
uniformly  colored,  but  show  a  distinct  polar  staining.  Frequently 
the  entire  periphery  is  stained  and  only  the  centre  left  uncolored. 
Other  forms,  differing  in  certain  respects  from  the  above  description 
and  not  representing  the  most  characteristic  type,  are  so  frequently 
found  in  smears  that  the  student  must  thoroughly  familiarize  himself 
with  them.  These  are  elliptical,  egg-shaped,  or  almost  spherical 
forms,  which  show  only  a  very  narrow  peripheral  staining,  or  do  not 
stain  at  all,  giving  the  appearance  of  empty  shells,  which  in  all  prob- 
ability is  the  case,  since  they  are  most  commonly  found  in  older  buboes. 
Various  involution  forms  are  also  frequently  seen  in  smears  from  cases 
which  have  succumbed  but  a  few  days  after  sickness.  They  appear 
like  yeast  cells,  and  are  either  quite  hazy  and  indistinct  or  club-shaped 
and  irregular  in  outline.  In  cover-glass  preparations  from  pure  cul- 
tures the  bacilli  are  not  so  characteristic.  Plague  bacilli  from  pure 
cultures,  particularly  from  the  water  of  condensation  of  agar  tubes,  or 
from  bouillon,  frequently  show  shorter  or  longer  chains,  in  which 
dividing  lines  between  the  individual  bacilli  are  so  indistinct  as  to 


236     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

cause  them  to  appear  like  filaments.  Involution  forms  are  also  liable 
to  present  themselves  early  in  a  similar  manner  even  on  favorable 
media. 

The  plague  bacillus  is  decolorized  by  Gram's  method.     When 
grown  in  the  animal  body  the  bacillus  possesses  a  capsule,  which, 

FIG.  127 


Postmortem  smear  from  the  spleen  in  a  case  of  bubonic  plague,  showing  polar  staining. 
(Author's  preparation.) 


FIG.  128 


FIG.  129 


Postmortem  smear  from  a  case  of  plague 
pneumonia,  showing  polar  staining. 
(Author's  preparation.) 


Cover-glass  preparation  from  a  twenty- 
four-hour-old  agar  plague  culture,  stained 
with  dilute  carbol-fuchsin,  showing  very 
small  bacilli,  with  rounded  ends.  (Author's 
preparation.) 


however,  is  difficult  to  demonstrate  unless  thin  spreads  are  prepared 
and  fixed  with  great  care  in  alcohol.  There  is  nothing  characteristic 
in  the  capsule,  so  its  exhibition  will  not  assist  in  the  microscopic 
diagnosis.  The  bacilli  are  not  motile,  do  not  possess  flagella,  nor 


BACILLUS  PESTIS 


237 


FIG.  130 


has  spore  formation  been  observed.  Even  though  bodies  somewhat 
resembling  spores  are  occasionally  seen  in  the  bacilli  they  are  not 
genuine  spores,  because  such  bacilli  are  not 
more  resistant  to  heat,  antiseptics,  etc.,  than 
the  other  pest  bacilli. 

Cultural  Properties. — The  plague  bacillus 
grows  on  all  ordinary  laboratory  culture 
media,  best  on  such  as  are  faintly  alkaline. 
Even  a  minor  degree  of  acidity  as  well  as 
a  higher  degree  of  alkalinity  prevents  devel- 
opment. It  develops  at  temperatures  ranging 
from  5°  to  38°  C.,  and  in  artificial  media  best 
at  25°  to  30°  C.  It  is  almost  strictly,  though 
perhaps  not  absolutely,  obligate  aerobic.  As 
a  rule,  it  develops  on  artificial  media  only  in 
the  presence  of  free  oxygen;  some  observers, 
however,  have  occasionally  seen  a  weak 
growth  in  its  absence.  When  a  favorable 
solid  culture  medium  (agar  or  gelatin,  slightly 
alkaline)  is  inoculated  from  the  organs  (bubo, 
spleen,  etc.)  of  a  plague  case  the  development 
of  the  bacilli  is  at  first  generally  quite  slow, 
and  frequently  very  little  can  be  seen  with  the 
naked  eye  in  the  first  twenty-four  hours. 
After  this  time  a  typical  picture  may  appear 
in  a  considerable  number  of  cases,  and  it  is 
always  present  after  forty-eight  hours.  The 


FIG.  131 


Culture  of  plague  bacillus 
on  agar,  five  days'  growth, 
fixed  with  formalin  vapor. 
(Author's  preparation.) 


Colony  of  plague,  gelatin  plate  culture,  forty-eight 
hours  old,  showing  dark  elevated  centre  and  trans- 
parent homogeneous  marginal  zone.  (Author's 
preparation.) 


surface  of  the  agar  or  gelatin  shows  small,  delicate,  round,  moist,  dew- 
drop-like  colonies.  They  are  light  gray  in  reflected  light  and  grayish 
white  in  transmitted  light.  If  these  colonies  are  inspected  with  a 
hand  lens  or  with  a  low  power  of  the  compound  microscope  they 
show  an  elevated,  finely  granular,  rounded  centre  and  a  perfectly 
transparent,  very  thin,  flat  marginal  zone.  The  colonies,  on  the  whole, 


238     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

are  circular,  but  the  transparent  marginal  zone  early  shows  a  some- 
what irregular  boundary  line.  If  a  young  plague  culture  is  touched 
with  a  platinum  loop  it  is  found  to  be  viscous  and  sticky.  It  is, 
however,  easily  removed  from  the  surface  on  which  it  grows. 

The  plague  bacillus,  as  already  stated,  has  a  marked  tendency  to 
develop  involution  forms  early  in  its  growth.  As  first  shown  by 
Hankin,  this  tendency  is  most  pronounced  in  cultures  on  a  3  to  4  per 
cent,  salt  agar,  one  of  the  most  valuable  media  for  the  bacteriologic 
diagnosis  of  plague.  It  is  prepared  and  standardized  like  an  ordinary 
agar,  except  that  it  contains,  instead  of  J  per  cent.,  3  to  4  per  cent,  of 
common  salt.  Generally  the  greater  number,  or  all,  of  the  organisms 
from  such  a  growth  present  themselves  as  large  spherical  bodies, 
looking  very  much  like  yeast  cells;  later,  large,  swollen  club-  or  dumb- 
bell-shaped or  irregular  forms  make  their  appearance.  The  most 
typical  and  most  constant  form  grown  on  a  3.5  to  4  per  cent,  salt 
agar,  after  twenty-four  to  forty-eight  hours,  is  the  yeast-like,  large, 
spherical  plague  organism.  No  other  microorganism  forms  this 
type  so  promptly  and  regularly  on  salt  agar  that  it  might  be  con- 
founded with  the  plague  bacillus.  Hence,  it  is  advisable  at  the  autopsy 
in  a  suspected  case  of  plague  to  inoculate  besides  gelatin  plates  also 
ordinary  agar  tubes,  bouillon  flasks,  and  salt-agar  tubes  or  plates. 
In  bouillon  flasks  the  bacilli,  at  temperatures  between  30°  to  35°  C., 
show  a  finely  flocculent  whitish,  slowly  increasing  sediment  after 
twenty-four  hours.  During  the  next  twenty-four  hours  the  flocculi 
extend  upward  from  the  bottom  along  the  walls.  A  fine  whitish  ring 
of  growth  then  forms  on  the  surface,  and  in  course  of  time  covers  it. 
If  the  flask  is  kept  motionless  and  undisturbed,  bands  and  strands  of 
bacilli  finally  grow  downward  from  the  surface  membrane.  The 
contents  of  the  flask  now  present  an  appearance  somewhat  resembling 
stalactite  and  stalagmite  formations. 

The  plague  bacillus  does  not  liquefy  gelatin  or  blood  serum;  does 
not  ferment  dextrose,  levulose,  lactose,  or  mannite;  and  grows  spar- 
ingly on  potato  and  in  milk.  It  does  not  coagulate  the  latter. 

Resistance. — The  resistance  of  the  plague  bacillus  is  not  great;  it 
is  about  the  same  as  that  shown  by  other  members  of  the  group.  At 
70°  C.  the  bacilli  are  safely  killed  within  one  hour;  sunlight  and  drying 
destroy  them  within  twenty-four  hours;  1  per  cent,  lysol  solution  in 
ten  to  fifteen  minutes;  1  per  cent,  caustic  lime  in  ten  minutes;  1  per 
cent,  corrosive  sublimate  solution  in  half  a  minute;  1  per  cent,  hydro- 
chloric acid  solution  in  one  minute;  J  per  cent,  sulphuric  acid  in  five 
to  ten  minutes. 

Vaccine  and  Serumtherapy. — Killed  cultures  of  the  plague  bacillus 
have  been  prepared  and  used  as  vaccines  by  Yersin,  Calmette,  Borrell, 
Haffkine,  and  others.  Haffkine's  has  been  used  extensively  in  India. 
Attenuated  but  live  cultures  have  been  used  by  Kolle  and  Otto  and 
Strong.  An  antiplague  serum  has  been  prepared  by  a  number  of 
investigators  by  the  inoculation  of  horses.  Vaccination  against  plague 


QUESTIONS  239 

with  dead  cultures  has  evidently  a  valuable  protective  influence;  the 
antiserum  has  not  been  very  successful  as  a  curative  agency  in  cases 
of  plague  after  they  are  sufficiently  well  developed  for  a  diagnosis. 

QUESTIONS. 

1.  What  is  a  hemorrhagic  septicemia?     What  are  its  most  characteristic 
pathologic  changes  or  lesions? 

2.  What  kind  of  bacteria  do  occasionally;  what  kind  generally  lead  to  hemor- 
rhagic septicemia? 

3.  Give  the  common  features  of  the  bacteria  of  the  group  of  Bacillus  pluri- 
septicus  (bipolaris). 

4.  Does  the  anthrax  bacillus  belong  to  this  group?    If  not,  why  not? 

5.  What  animal  diseases  are  due  to  bacilli  of  the  hemorrhagic  septicemia 
group?    What  have  they  been  called  by  the  French  writers?    What  is  meant 
by  the  term  Paste  urella? 

6.  Why  are  these  organisms  known  as  the  Bacillus  bipolaris  septicus? 

7.  What  was  Pasteur's  work  in  connection  with  the  disease  known  as  chicken 
cholera? 

8.  Describe  the  pathologic  lesions  of  this  disease. 

9.  In  what  part  of  the  intestines  are  the  changes  most  pronounced? 

10.  Describe  the  morphology  and  cultural  properties  of  the  Bacillus  (bipolaris) 
avisepticus. 

11.  How  can  its  virulency  be  increased;  how  can  the  organism  be  attenuated? 

12.  What  animals  are  susceptible  to  the  organisms? 

13.  What  steps  are  necessary  in  establishing  beyond  doubt  the  diagnosis  of 
fowl  cholera? 

14.  Discuss  the  resistance  of  the  Bacillus  avisepticus  and  the  effective  methods 
of  disinfecting  infected  hen  houses. 

15.  How  can  active  and  passive  immunity  against  fowl  cholera  be  procured? 

16.  Does  the  fowl  cholera  bacillus  form  a  soluble  toxin? 

17.  Where  does  hemorrhagic  septicemia  of  cattle  occur? 

18.  Describe   its  pathologic   lesions.     What  are    the   characteristics  of  the 
spleen  in  hemorrhagic  septicemia  and  in  anthrax? 

19.  Describe  the  morphologic  and  cultural  properties  of  the  Bacillus  (bipolaris) 
bovisepticus. 

20.  Is  the  cornstalk  disease  of  cattle  due  to  this  bacillus? 

21.  What  is  the  cause  of  septic  pleuropneumonia  of  calves? 

22.  WTiat  is  the  cause  of  hemorrhagic  septicemia  in  sheep? 

23.  Why  has  there  been  a  considerable  confusion  in  the  past  as  to  the  etiology 
and  pathology  of  hemorrhagic  septicemia  of  swine? 

24.  Give  the  other  names  for  this  disease. 

25.  What  organism  is  the  cause  of  swine  plague? 

26.  Describe  the  most  prominent  pathologic  lesions  in  a  very  acute  case  of 
swine  plague. 

27.  What  is  the  meaning  of  the  terms  petechia?  and  ecchymoses? 

28.  What  are  the  most  prominent  pathologic  lesions  in  chronic  swine  plague? 

29.  Describe  the  Bacillus  (bipolaris)  suisepticus.    Where  is  it  found  in  acute, 
where  in  chronic  cases? 

30.  What  is  the  vacuole  bacillus  of  Bang?     What  the  Bacillus  parvus  ovatus 
of  Loeffler? 

31.  Describe   the   cultural   properties  of   the   swine  plague   bacillus.     What 
animals  are  susceptible  to  it? 

32.  Discuss  the  resistance  of  the  organism. 

33.  What  is  a  polyvalent  immune  or  antitoxic  serum? 

34.  How  is  the  polyvalent  antiswine  plague  serum  of  Ostertag-Wassermann 
prepared  ? 

35.  Under  what  other  names  is  the  equine  disease  pink-eye  known? 

36.  What  organism  according  to  Ligmere  causes  this  disease? 

37.  Describe  the  most  prominent  lesions  of  an  acute  case  of  equine  influenza. 

38.  What  are  the  principal  lesions  in  the  pectoral  form  of  the  disease? 

39.  Describe  the  morphology  and  cultural  properties  of  the  Bacillus  equi- 
septicus  of  Ligniere. 


240     BACILLI  OF  THE  HEMORRHAGIC  SEPTICEMIA  GROUP 

40.  What  animals  are  susceptible  to  the  pathogenic  action  of  the  bacillus; 
how  does  natural  infection  occur? 

41.  Discuss  the  canine  disease  known  as  dog  typhus  or  gastro -enteritis  hemor- 
rhagica. 

42.  What  organism  causes  bubonic  plague  in  man,  rats,  and  other  animals? 

43.  Define  and  describe  a  plague  bubo. 

44.  Define  the  following  terms:    primary  bubo  of  the  second  order;  secondary 
bubo  of  the  first  order. 

45.  What  lymph  glands  in  rats  are  most  commonly  the  seat  of  the  primary 
bubo? 

46.  Name  the  lymph  glands  affected  in  the  order  of  their  frequency. 

47.  Describe  the  liver  changes  frequently  found  in  rats  dead  from  plague 
infection. 

48.  Describe   the   appearance   of  the   subcutaneous   connective   tissue   after 
removal  of  the  skin. 

49.  What  pathologic  change  in  the  pleura  is  very  typical  for  rat  plague? 

50.  What  other  animals  in  general  are  said  to  be  susceptible  to  natural  plague 
infection?    What  other  rodents  in  addition  to  the  rat  are  susceptible? 

51.  How  is  plague  infection  spread  from  rat  to  rat  and  from  rat  to  man? 

52.  Is  the  general  transmission  by  rat  fleas  proved  beyond  doubt?    If   not, 
why  not? 

53.  Discuss  the  finding  of  plague  bacilli  in  plague -sick  rats. 

54.  Is  plague  in  man  generally  a  true  septicemia? 

55.  What  is  latent  plague?    Among  what  animals  or  human  beings  does  it 
occur? 

56.  Describe  the  morphology  and  staining  properties  of  the  Bacillus  pestis. 

57.  Describe  its  cultural  properties. 

58.  Discuss  its  resistance. 

59.  Have  vaccine  and  serumtherapy  been  tried  in  plague,  and  how? 


CHAPTEE    XX. 

ANTHRAX  BACILLUS. 

Occurrence  and  Pathogenesis. — Anthrax,  splenic  fever,  malignant 
pustule,  carbuncle,  woolsorter's  disease,  Milzbrand  (German),  char- 
bon  (French),  is  a  disease  of  man  and  the  lower  animals  which  has 
apparently  been  known  to  mankind  for  thousands  of  years.  It  is 
probably  the  affection  referred  to  in  the  Bible  (Exodus,  ix,  3-9),  and 
it  is  mentioned  by  several  of  the  classic  Greek  writers,  including 
Homer.  Its  names  are  derived  from  the  most  obvious  anatomic 
lesions,  enlargement,  softening,  and  congestion  of  the  spleen  (splenic 
fever,  Milzbrand),  and  the  widespread  occurrence  of  acute  passive 
congestion  and  acute  hemorrhagic  infiltration  of  the  subcutaneous, 
subserous,  and  submucous  tissues,  which  generally  give  to  them  a  very 
dark  brown  or  dark  purple  or  sometimes  an  almost  black  color.  The 
term  anthrax  is  derived  from  the  Greek  word  for  coal,  which  is  also 
the  meaning  of  the  French  charbon.  The  designation  woolsorter's 
disease  is  derived  from  the  fact  that  the  handlers  of  hides  from  cows 
or  wool  from  sheep  dead  from  anthrax  often  contracted  the  disease, 
which  in  this  case  usually  assumes  the  local  character  of  a  malignant 
pustule  or  carbuncle  or  an  inhalation  pulmonary  affection.  The  disease 
is  caused  by  a  pathogenic  microorganism  known  as  the  anthrax 
bacillus.  It  is  most  common  among  cattle  and  sheep,  but  also  occurs 
in  man,  horses,  hogs,  goats,  deer,  hares,  buffaloes,  dogs,  cats,  and  very 
rarely  among  chickens,  ducks,  and  geese.  An  anthrax  epidemic  among 
the  wild  animals  of  the  zoological  garden  of  Copenhagen  has  been 
described  by  Jensen,  and  a  laboratory  epidemic  among  guinea-pigs 
that  contracted  the  disease  from  anthrax-infected  peat  has  also  been 
reported.  The  susceptible  laboratory  animals  used  in  experimental 
work  are  mice,  guinea-pigs,  and  rabbits.  Gray  rats  are  very  slightly 
susceptible,  white  rats  more  so.  Anthrax  is  prevalent  particularly 
among  cattle  and  sheep  practically  throughout  the  entire  world.  It 
has  been  found  in  Europe,  Asia,  Africa,  Australia,  and  North  and 
South  America,  but  it  is  by  no  means  equally  distributed  in  all  places, 
being  much  more  prevalent  in  moist,  marshy  lowlands  or  prairies 
than  in  dry,  rocky  soil.  It  is  endemic  in  favorable  localities,  where  it 
finds  its  numerous  victims  year  after  year;  it  also  occurs  in  sporadic 
outbreaks. 

Pathologic  Lesions. — The  anatomical  changes  of  anthrax  are  quite 
characteristic.  The  blood,  on  account  of  a  lack  of  oxygenation  due 
to  toxic  influences,  becomes  very  dark  and  does  not  promptly  and 
16 


242  ANTHRAX  BACILLUS 

sufficiently  coagulate  after  death.  Postmortem  rigidity  is  not  strong 
nor  does  it  continue  very  long,  and  putrefactive  changes  with  gas 
formation  rapidly  set  in.  Blood  oozes  from  the  mouth,  the  nose, 
and  the  anus.  The  mucous  and  serous  membranes  are  enormously 
congested  and  show  petechice  and  ecchymoses.  Hemorrhages  in  the 
connective  tissue  are  also  found  in  various  places.  The  subcutaneous 
and  intramuscular  connective  tissues  show  an  cedematous,  gelatinous 
infiltration  with  hemorrhagic  patches  here  and  there.  The  lymph 
glands  are  swollen  and  edematous.  The  spleen  is  generally  much 
swollen,  much  congested,  its  pulp  very  soft,  and  the  capsule  tense. 
It  ruptures  easily,  and  an  almost  fluid  pulp  mass  may  be  discharged 
spontaneously.  The  liver  and  kidneys  are  swollen,  congested,  and, 
when  cut  into,  discharge  much  dark  blood  and  present  a  dull  surface 
indicating  parenchymatous  degeneration.  The  lungs  and  the  brain 
are  likewise  hyperemic  and  edematous.  The  mucosa  of  the  intestines 
is  swollen,  dark,  sometimes  necrotic,  often  raised  in  patches  by 
edematous  and  hemorrhagic  infiltration  in  the  form  of  ridges  or 
globular  masses.  The  blood,  when  examined  microscopically,  shows 
a  very  marked  increase  in  the  number  of  leukocytes  (leukocytosis  or 
hyperleukocytosis)  and  enormous  numbers  of  anthrax  bacilli.  These, 
however,  are  found  in  the  capillaries  of  the  internal  organs  rather 
than  in  the  larger  vessels.  Sometimes  in  very  rapid  so-called  ful- 
minant cases  the  anatomical  changes  are  not  as  well  marked  as 
described  above,  because  the  fatal  intoxication  has  been  so  rapid  that 
the  characteristic  anatomical  changes  have  not  had  sufficient  time  to 
develop.  The  spleen,  however,  is  generally  very  congested,  enlarged 
and  softened  in  all  cases. 

The  Discovery  of  the  Bacillus. — Anthrax  bacilli  were  first  seen  in 
the  blood  of  animals  dead  from  the  disease  by  Pollender  and  Brauell. 
The  latter  also  made  some  successful  inoculation  experiments  on 
animals.  These  were  later  repeated  and  extended  by  Davaine,  who 
had  previously  written  concerning  the  rod-shaped  bodies  in  the  blood 
of  animals  which  had  died  from  splenic  fever.  The  etiology  of  anthrax 
was  first  firmly  established  by  pure  culture  and  animal  experiments 
by  Robert  Koch,  in  1876,  who  was  also  the  first  to  discover  the  spores 
of  the  bacillus. 

Morphology. — The  Bacillus  anthracis  is  a  rod-shaped  bacterium 
1.5  to  10  micra  long  (average  length,  4  micra),  and  1  to  1.25  micra 
thick.  It  is,  therefore,  one  of  the  largest  pathogenic  bacteria.  It  is  not 
motile.  The  exact  morphology  of  the  bacilli  can  best  be  studied  in 
the  blood  of  animals  which  have  just  died  or  are  in  the  agonal  stage 
of  the  disease.  The  bacilli  appear  in  large  numbers  in  the  blood  ten  to 
twelve  hours  before  death.  Examination  of  the  unstained  blood  of  a 
mouse  or  guinea-pig  in  a  moist  cover-glass  preparation  shows  trans- 
parent, colorless,  cylindrical  rods  which  have  no  motility  whatever 
between  the  erythrocytes.  Individual  bacilli  or  short  chains  of  two, 
three,  or  four  bacilli  occur.  Long  chains  or  spores  are  never  seen  in 


PLATE  VI 


Bacillus  of  Anthrax  in  the  Blood  of  a  Cow. 

Natural  infection.     Capsules  stained  with  eosin. 


SPORES  243 

fresh  blood  preparations.  If  the  blood  or  the  juice  from  the  spleen 
is  obtained  a  considerable  time  after  death,  when  putrefactive  changes 
and  the  admission  of  air  has  set  in,  longer  chains  and  spores  may  be 
found. 

Staining  Properties. — The  anthrax  bacillus  stains  well  with  the 
ordinary  watery  anilin  stains,  and  is  Gram  positive.  When  fixed  and 
stained  in  the  usual  manner  the  bacilli  show  certain  features  not  seen 
in  the  fresh,  unstained  specimen.  The  ends  of  the  bacilli  appear 
sharper  and  are  not  so  regularly  cylindrical  as  before,  but  slightly 
swollen,  and  show  a  little  excavation  of  the  outer  surface,  so  that  a 
small  lenticular  empty  space  is  formed  between  two  individual  bacilli 
of  a  chain.  A  chain  of  anthrax  bacilli  after  it  is  stained  looks  very 
much  like  a  stick  of  bamboo.  In  the  stained  blood  or  splenic  juice 
preparation  the  anthrax  bacillus  shows  a  gelatinous  capsule,  which, 
however,  is  not  present  in  bacilli  obtained  from  pure  cultures  on  the 
ordinary  artificial  media.  Johne  has  devised  a  special  method  for 
clearly  demonstrating  the  anthrax  bacillus  capsule  in  blood  prepa- 
rations. His  method  is  as  follows: 

1.  Prepare  a  blood  smear  on  a  cover-glass,  allow  it  to  become  air 
dry,  and  fix  by  drawing  carefully  and  rapidly  through  a  flame.    Do 
not  overheat  or  the  capsules  will  be  burned. 

2.  Apply  to  the  fixed  cover-glass  2  per  cent,  watery  gentian-violet 
solution  and  heat  slightly  for  one-quarter  to  one-half  minute  over  a 
flame. 

3.  Wash  rapidly  in  water. 

4.  Apply  for  six  to  ten  seconds  a  1  to  2  per  cent,  solution  of  acetic 
acid. 

5.  Wash  in  water  and  mount  in  water  (not  in  Canada  balsam)  on 
a  slide  and  examine  while  moist. 

The  capsules  may  also  be  stained  by  any  of  the  other  methods  for 
exhibiting  this  plasmatic  envelope. 

Spores. — Spores  can  easily  be  demonstrated  in  cultures  raised  on 
artificial  culture  media  in  the  presence  of  oxygen.  As  far  as  is  known, 
the  organism  never  forms  spores  in  the  absence  of  free  oxygen,  and 
they  are  never  seen  in  the  living  blood.  They  are  formed  in  artificial 
media  at  blood  temperature  after  eighteen  hours,  at  18°  C.,  after 
fifty  hours;  at  14°  C.  spores  are  no  longer  formed.  The  spore  of  the 
anthrax  bacillus  is  found  in  the  middle  of  the  rod,  but  since  the  proto- 
plasm of  the  latter  soon  degenerates  and  perishes  after  sporulation, 
it  may  appear  as  if  this  were  not  the  case.  Every  bacillus  forms  a 
single  spore,  which  is  not  perfectly  spherical  but  somewhat  egg-shaped 
or  elliptical.  These  spores  possess  a  very  tough  membrane;  some 
observers  even  claim  that  the  anthrax  spore  has  two  membranes. 
The  following  method  of  staining  the  spores  is  recommended  as  the 
most  trustworthy: 

1.  Take  several  platinum  loopfuls  of  material  from  an  anthrax 
growth  on  a  solid  medium  and  rub  it  up  well  with  a  0.85  per  cent, 
salt  solution. 


244 


ANTHRAX  BACILLUS 


FIG.  132 


2.  Mix  1  or  2  c.c.  of  this  emulsion  with  an  equal  amount  of  Ziehl's 
carbol  fuchsin  and  place  in  a  watch-glass  or  small  beaker. 

3.  Heat  over  a  small  flame  for  about  six  minutes,  until  vapor  rises. 

4.  Spread  some  of  the  mixture  upon  a  cover-glass  or  slide,  allow  the 
preparation  to  become  air  dry,  and  fix  by  drawing  twice  through  a  flame. 

5.  Decolorize  for  one  or  two  seconds  in  1  per  cent,  sulphuric  acid 
watery  solution;   wash   in  water,  counterstain   in  watery  methylene 
blue  for  three  or  four  minutes. 

As  a  result  of  the  stain  the  spores  show  red  and  the  bacilli  blue. 
The  best  culture  medium  for  the  production  of  an  abundant  spore 

formation  is  boiled  sterilized 
potato,  cut  into  halves  or  disks, 
and  kept  at  temperatures  be- 
tween 31°  and  37°  C.  Another 
excellent  method  for  obtaining 
abundant  sporulation  consists  in 
pouring  bouillon  containing 
anthrax  bacilli  on  agar  plates. 
When  spore  formation  is  studied 
directly  under  the  microscope 
the  following  changes  are  seen. 
There  first  occurs  a  clouding 
of  the  previously  clear  proto- 
plasm of  the  bacillus,  the  latter 
forming  fine  granules  and  ap- 
pearing as  if  it  had  been  dusted. 
Then  larger,  highly  refractive 
granules  appear,  and  these, 
probably  by  uniting  with  each 
other  and  with  other  substances  of  the  bacillus,  form  the  round  or 
slightly  oval  spore. 

Germination  of  Spores. — Spores  or  sporulating  bacilli  under  favor- 
able conditions,  either  in  nature,  in  the  body  of  a  susceptible  animal, 
or  in  a  suitable  culture  medium,  develop  again  into  young  bacilli. 
The  tough  spore  membrane  swells  up  and  becomes  elongated,  the 
spore  itself  within  the -membrane  loses  its  shining  refractive  appear- 
ance, and  a  hole  or  rupture  appears  in  the  membrane  through  which 
the  elongated  spore,  having  now  assumed  the  shape  of  a  young  bacillus, 
makes  its  exit.  Occasionally  a  short  bacillus  with  a  round,  empty 
capsule  at  one  end  is  seen  in  preparations  from  anthrax  cultures, 
giving  the  appearance  of  a  bacillus  with  a  spore  at  one  pole,  such  as 
is  found  in  the  case  of  the  sporulating  tetanus  bacillus.  With  the 
anthrax  bacillus,  however,  this  appearance  is  deceptive  and  in  reality 
only  indicates  a  young  bacillus  and  an  empty  spore  membrane  or 
capsule  at  one  end.  Germination  of  spores,  after  they  have  been 
brought  into  a  favorable  culture  medium,  and  if  kept  at  temperatures 
between  30°  and  37°  C.,  begins  generally  after  eight  hours. 


Anthrax  bacillus,  spore  formation     X   1000. 
(Author's  preparation.) 


CULTURAL  PROPERTIES 


245 


Asporogenous  Anthrax  Bacillus. — A  variety  of  the  anthrax  bacillus 
which  no  longer  forms  spores  may  be  raised  by  adding  certain  anti- 
septics, such  as  carbolic  acid  in  the  proper  proportion  to  the  culture 
medium.  After  these  artificially  produced  varieties  have  once  lost  the 
property  of  forming  spores,  it  is  not  regained  even  when  theyjire 
transplanted  to  culture  media  free  from  antiseptics. 

FIG.  133 


Six  cultures  of  anthrax  bacillus,  showing  increase  in  liquefaction,  with  age. 

preparation.) 


(Author's 


Cultural  Properties. — Artificial  cultivation  of  the  anthrax  bacillus 
is  very  easy.  It  can  be  accomplished  from  infected  blood,  discharges 
or  tissues  by  the  plate  method  or  by  preliminary  inoculation  of  the 
infected  material  into  mice  or  guinea-pigs.  The  organism  grows  well 
on  all  the  ordinary  laboratory  culture  media.  It  has  a  wide  range  of 
temperature  (between  15°  and  43°  C.),  in  which  it  can  grow  and 
multiply,  the  most  favorable  temperature  being  between  30°  and  40°  C. 
It  not  only  grows  well  on  the  ordinary  laboratory  media,  but  also  on 
sterilized  disks  of  cucumbers,  in  the  sterilized  and  neutralized 
juice  of  pears,  onions,  and  beets;  in  urine,  provided  it  is  not  too 
alkaline,  and  particularly  well  in  urine  containing  albumin.  It  is 
readily  seen  that  an  organism  thriving  at  such  a  great  range  of  tem- 
perature and  in  so  great  a  variety  of  media  easily  finds  opportunity 
to  multiply  in  the  outside  world  as  a  saprophyte. 

Gelatin. — On  gelatin  plates  the  organism  first  forms  small,  round, 
grayish-white  colonies  upon  the  surface;  these  rapidly  begin  to  liquefy 
the  culture  soil.  In  the  depth  of  the  medium  the  young  colonies  are 
seen  as  elliptical,  slightly  brownish,  granular,  small  points  or  disks, 


246  ANTHRAX  BACILLUS 

which  do  not  grow  as  well  as  the  surface  colonies.  The  latter  soon 
spread  out  into  larger  spots,  with  a  still  generally  spherical  but  more 
irregular  outline,  from  which  bundles  of  filaments  and  cords  formed 
by  the  bacilli  project.  The  whole  formation  then  somewhat  resembles 
a  tuft  of  wool  or  the  serpent-covered  head  of  the  mythological  Greek 
Medusa,  as  can  be  demonstrated  in  stained  specimens,  prepared  by 
the  so-called  impression  method  (Klatsch-Praparat,  German).  Such 
preparations  are  made  by  pressing  a  perfectly  clean  dry  cover-glass 
upon  a  surface  colony  on  a  gelatin  plate  and  then  carefully  lifting  it  off 
with  forceps.  The  impression  on  the  cover-glass  is  then  air  dried, 
fixed,  and  stained  in  the  usual  manner. 

FIG.  134 


Colonies  of  bacillus  anthracis  upon  gelatin  plates:  a,  at  the  end  of  twenty-four  hours;  b,  at  the 
end  of  forty-eight  hours.      X  80.     (F.  Fliigge.) 

A  gelatin  stick  culture  of  anthrax  is  very  characteristic.  The  growth 
first  develops  on  the  surface  and  along  the  stab,  because  the  bacillus 
requires  oxygen  in  artificial  cultures.  The  very  surface  growth,  for 
this  reason,  is  most  luxuriant,  and  the  pellicle  formed  is  irregular 
and  uneven.  Along  the  stab  itself  a  radiating  or  arborescent  effect  is 
produced,  making  the  culture  at  first  somewhat  resemble  an  inverted 
pine  tree.  Liquefaction  of  the  gelatin  is  most  pronounced  at  the 
surface  where  the  growth  is  most  abundant.  Some  of  the  liquefied 
material  runs  down  into  the  canal  of  the  stab  and  some  of  the  fluid 
is  lost  by  evaporation,  causing  a  cup-like  or  cone-  or  funnel-shaped 
depression  to  form  on  the  surface.  After  several  days  when  the  growth 
has  become  more  abundant  the  entire  upper  stratum  of  the  gelatin 
may  be  liquefied  and  the  growth  itself  sinks  to  the  bottom  of  the 
fluid  layer. 

Agar  and  Other  Media. — On  agar  plates  the  development  is  similar 
to  that  on  gelatin  plates.  There  is,  of  course,  no  liquefaction,  because 


RESISTANCE  OF  THE  ANTHRAX  BACILLUS  AND  ITS  SPORES     247 

the  peptonizing  ferment  of  bacteria  digests  and  fluidifies  gelatin, 
blood  serum,  egg-albumen,  etc.,  but  not  agar.  Agar  stick  cultures 
also  resemble  gelatin  stick  cultures,  except  for  the  absence  of  lique- 
faction, and  the  inverted  pine-tree  effect  is  often  well  produced.  On 
agar  slants  gray,  granular,  shiny  growths  develop,  which  later  become 
uneven  and  folded  and  cannot  be  very  easily  removed  from  the  surface 
for  microscopic  examination  without  the  use  of  some  force  with  the 
platinum  loop.  On  potatoes  the  anthrax  bacillus  grows  very  luxuri- 
antly, forming  a  whitish,  dull,  shining  mass,  and  the  spore  formation 
is  generally  very  abundant  and  seen  early.  The  growth  on  blood 
serum,  as  on  gelatin,  leads  to  liquefaction.  Sterile  milk  is  first  coagu- 
lated by  the  anthrax  bacillus,  later  the  precipitated  casein  is  liquefied. 
Fresh  unpasteurized  or  unsterilized  milk  is  not  a  favorable  culture 
soil,  because  the  developing  lactic  acid  bacteria  prevent  the  growth 
of  anthrax  bacilli  causing  them  very  soon  to  die  out  completely, 
unless  spores  have  previously  been  formed.  In  bouillon  the  growth 
of  the  heavy  non-motile  bacilli  leads  to  the  formation  of  slimy  flocculi 
which  sink  to  the  bottom.  From  this  sediment  filamentous  masses 
arise,  but  the 'upper  layers  of  the  fluid  generally  remain  clear. 

Resistance  of  the  Anthrax  Bacillus  and  its  Spores. — The  vegetative 
form  of  the  anthrax  bacillus  is  not  very  resistant  to  inimical  germi- 
cidal  and  physical  influences,  but  the  spores  are  much  more  resistant 
than  those  of  most  other  pathogenic  bacteria;  tetanus  spores, 
however,  withstand  moist  heat  better  than  anthrax  spores.  Anthrax 
bacilli  are  killed  when  heated  to  55°  C.  for  forty  minutes  if  contained 
in  fresh  blood,  and  in  one  hour  if  heated  to  50°  C.  A  spore-free 
bouillon  culture  is  killed  if  exposed  for  five  and  one-half  minutes  to 
65°  C.,  and  in  three  minutes  if  heated  to  75°  C.  Dry  air  of  140°  C. 
kills  anthrax  spores  only  after  three  hours'  exposure;  steam  of  95°  C. 
after  ten  minutes;  steam  of  100°  C.  after  five  minutes.  Low  tempera- 
tures as  they  ordinarily  occur  in  nature  in  countries  of  medium 
latitude  have  no  appreciable  effect  upon  either  bacilli  or  spores. 
Direct  sunlight  kills  bacilli  and  spores  in  a  comparatively  short  time, 
particularly  when  the  air  has  free  access  while  the  rays  are  acting 
upon  the  organism.  Bacilli  are  easily  killed  by  a  1  to  1000  solution  of 
corrosive  sublimate,  and  even  spores  are  killed  after  a  few  hours, 
but  this  powerful  action  is  only  obtained  when  the  solutions  act  upon 
dry  bacilli  or  spores  or  when  the  latter  are  suspended  in  a  watery 
solution.  In  the  presence  of  albumin,  as  in  blood,  the  action  of  mer- 
cury bichlorid  is  untrustworthy.  Carbolic  acid  in  watery  solutions 
acts  weakly  toward  spores.  Salting  and  curing  of  meat  kill  anthrax 
bacilli  in  from  two  to  four  weeks,  but  has  no  effect  upon  the  spores. 
The  most  markedly  antagonistic  bacterium  to  the  anthrax  bacillus 
is  the  Bacillus  pyocyaneus.  If  a  mixed  culture  of  anthrax  and 
pyocyaneus  is  made  the  latter  will  develop  and  the  former  will  die 
out.  This  effect  is  probably  due  to  a  ferment  secreted  by  the 
Bacillus  pyocyaneus,  and  known  as  pyocyanase.  The  streptococcus 


248  ANTHRAX  BACILLUS 

and  staphylococcus  are  also  antagonistic  to  anthrax,  but  to  a  less 
degree. 

Modes  of  Infection  under  Natural  Conditions. — Robert  Koch  has  done 
more  than  anyone  else  to  show  how  anthrax  is  spread  by  the  secretions 
of  sick  animals  and  from  cadavers.  The  excreta  and  the  hemorrhagic 
discharges  from  the  mouth,  nose,  and  anus  contain  anthrax  bacilli, 
which  are  likely  to  invade  barns  and  pastures.  Bacilli  are  also  spread 
when  cadavers  are  skinned  and  opened  and  when  they  are  buried  super- 
ficially. Spore  formation  occurs  under  all  these  conditions.  While 
anthrax  bacilli  themselves  easily  perish  in  the  presence  of  numerous 
putrefactive  bacteria,  spores  when  once  formed  are  very  resistant 
and  can  survive  in  moist  and  dry  soil  for  two  or  three  years,  in  water 
for  seventeen  months,  and  in  cesspools  for  fifteen  months.  After 
spores  have  formed  they  may  be  spread  by  infected  hides  and  by  hay 
collected  from  infected  pastures  and  stored  in  barns.  Ravenel  was  able 
to  show  that  anthrax  was  spread  by  a  tannery  working  with  infected 
hides;  others  have  demonstrated  the  persistence  of  anthrax  spores  for 
twelve  to  twenty  years  in  sand  in  which  an  anthrax  cadaver  had  been 
superficially  buried.  Kitasato,  on  the  other  hand,  demonstrated  that 
there  is  no  spore  formation  in  an  infected  cadaver  buried  as  deep  as 
six  feet.  By  far  the  most  common  route  of  infection  is  by  the  ingestion 
of  food  contaminated  with  anthrax  spores.  The  vegetative  form,  i.  e., 
the  bacillus,  when  taken  into  the  healthy  stomach,  is  probably  always, 
at  least  generally,  destroyed  by  the  gastric  juice;  but  the  spores  pass 
the  stomach  and  germinate  in  the  intestines,  where  the  bacilli  multiply 
and  break  through  the  mucous  membrane  into  the  general  circulation. 
Domestic  animals  also  show  an  inoculation  anthrax  through  wounds 
of  the  buccal  mucosa  or  skin.  Pulmonary  inhalation  anthrax  prob- 
ably does  not  occur  among  domestic  animals.  Intestinal  anthrax  is 
not  always  contracted  in  the  pasture,  and  may,  during  winter,  be 
contracted  from  stored  infected  hay. 

It  has  been  observed  that  anthrax  sometimes  occurs  sporadically 
in  a  single  animal  or  a  few  individuals  of  a  herd  while  the  great 
majority  escape  entirely.  Sobernheim  believes  this  to  be  due  to  a 
mild  unnoticed  form  of  anthrax  common  among  cattle  and  sheep  in 
anthrax-infected  neighborhoods,  which  leads  to  immunization  of  the 
majority  of  the  herd  by  a  natural  mild  infection. 

Anthrax  in  man  occurs  chiefly  in  persons  who  are  exposed  to  the 
disease  by  means  of  infected  hides,  wool,  and  other  similar  carriers. 
The  most  common  form  is  the  local  wound  infection,  the  anthrax 
carbuncle;  the  inhalation  pulmonary  form  follows,  and  the  ingestion 
intestinal  form  is  least  common.  The  cutaneous  form  usually  ends  in 
recovery,  while  the  pulmonary  and  intestinal  forms,  as  a  rule,  lead 
rapidly  to  a  fatal  termination. 

Diagnosis. — While  the  clinical  symptoms  and  anatomical  findings 
in  anthrax  are  quite  characteristic,  they  more  or  less  resemble  those 
of  emphysematous  anthrax  or  black-leg,  malignant  edema,  hemor- 


PLATE  VII 


i^v^SS&B&i 

*&&&&&'**$&  . 

,\^.«^v  %„•>•      ' 

"**$fifr  v**'  '' 


Kidney  of  Steer.    Anthrax  Bacillus  Infection. 

Zeiss  objective  4  mm.;  compens.  ocular  No.  6. 


DIAGNOSIS  249 

rhagic  septicemia,  etc.,  and  for  this  reason,  microscopic  and  laboratory 
examinations  are  necessary  for  a  trustworthy  diagnosis.  This  is 
particularly  necessary  in  the  case  of  a  fresh  outbreak  or  when 
the  disease  appears  in  a  district  which  has  previously  been  free  from 
anthrax.  Sufficient  evidence  for  a  trustworthy  diagnosis  can  often 
be  obtained  from  a  microscopic  examination  of  the  blood,  particularly 
in  the  last  stages  of  the  disease  while  the  animal  is  still  alive  or  within 
a  few  hours  after  death,  before  the  body  has  been  opened.  In  the 
former  case  blood  may  be  obtained  from  the  ear  by  a  slight  cutaneous 
incision.  When  a  postmortem  examination  is  made  it  is  best  to  take 
the  blood  from  the  spleen.  Blood  obtained  in'this  way  from  the  small 
peripheral  vessels  or  the  spleen  may  show  the  non-motile,  sporeless, 
cylindrical,  square-ended,  capsule-possessing  bacilli  in  short  chains. 
When  putrefaction  sets  in,  putrefactive  bacteria  which  closely  resemble 
the  anthrax  bacillus  appear  in  the  cadavers  of  cattle  and  sheep. 
Under  unfavorable  conditions,  cultures  must  therefore  be  prepared 
or  animal  experiments  made.  When  there  is  access  to  neither  a 
microscope  nor  laboratory  facilities  in  cases  of  suspected  anthrax  in 
cattle  and  sheep,  the  material  should  be  prepared  for  subsequent 
examination  in  a  laboratory  in  the  following  manner: 

1.  Prepare  a  few  microscopic  slides  by  spreading  some  blood  from 
the  animal  on  them  in  thin  layers. 

2.  Clean  a  wide-mouthed  bottle  by  washing  it  with  an  antiseptic 
solution  and  then  with  alcohol.    Invert  the  bottle  and  allow  it  to  dry. 
Place  in  it  a  portion  of  spleen,  or  if  the  cadaver  has  not  been  opened, 
a  portion  of  the  ear,  and  close  the  bottle  tightly  with  a  cork,  the  inner 
end  of  which  has  been  sterilized  by  burning. 

3.  Take  a  potato,  clear  it  of  earth,  wash  and  scrub  it  externally 
with  a  1  to  1000  solution  of  corrosive  sublimate.    Boil  it  well  in  water 
in  any  suitable  covered  vessel.    After  the  water  has  cooled,  take  out 
the  potato,  dry  it  externally  with  a  clean  cloth.    Cut  it  into  halves  with 
a  knife  previously  heated  over  a  flame.    In  this  manner  a  perfectly 
sterile  potato  is  obtained.     Allow  some  blood  to  flow  on  one  of  the 
flat  surfaces,  place  the  halves  again  face  to  face,  and  wrap  up  the 
potato,  if  possible,  in  clean  aseptic  gauze.  Place  the  potato  in  a  suitable 
container  (glass  vessel,  clean  tin  cup,  empty  gauze  box,  etc.). 

4.  Send  the  blood  slides,  piece  of  spleen,  and  inoculated  potato  to 
the  laboratory  by  the  quickest  possible  route  without  loss  of  time. 

The  trained  bacteriologist  is  immediately  able  to  stain  and  micro- 
scopically examine  the  blood  slides.  He  can  soon  examine  the  culture 
on  the  potato  and  from  it  and  the  piece  of  spleen  inoculate  mice  and 
guinea-pigs.  These  animals  are  very  susceptible,  and  will  show,  in 
one  or  two  days,  a  typical  anthrax  infection  if  the  material  actually 
contained  anthrax  bacilli.  The  inoculation  of  the  suspected  material 
is  made  subcutaneously  from  an  emulsion,  or  a  piece  is  introduced 
into  a  small  subcutaneous  pocket,  made  with  a  slender  scalpel  or  a 
pair  of  small  scissors.  Mice  and  guinea-pigs  infected  in  this  manner 


250  ANTHRAX.  BACILLUS 

soon  become  quiet,  refuse  to  take  food,  and  after  twenty-four  to  forty- 
eight  hours  suddenly  fall  over  with  some  convulsions  and  die.  Death 
is  due  to  paralysis  of  the  respiratory  centres  and  asphyxia.  The  post- 
mortem examination  shows  an  edematous  gelatinous  infiltration  at 
the  place  of  inoculation;  a  congested,  soft,  and  very  much  enlarged 
spleen;  and  dark  blood  which,  under  the  microscope,  exhibits  the 
bacilli  as  described  above.  These  findings  make  the  diagnosis  of 
anthrax  absolute. 

Prophylaxis. — Prophylaxis  against  the  spreading  of  anthrax  should 
in  the  first  place  concern  cadavers.  The  best  method  of  disposal  is 
to  bury  them  in  quicklime  at  or  near  the  place  where  the  animals 
have  died  without  loss  of  time  and  without  opening  or  skinning  them. 
The  bodies  should  be  interred  at  a  depth  of  five  to  six  feet,  so  that 
they  may  be  completely  covered.  Sick  animals  in  which  there  is 
little  prospect  of  recovery  should  be  killed,  but  not  by  bleeding,  and 
the  cadaver  should  be  treated  as  recommended  above.  The  place  of 
burial  should  be  fenced  in  for  several  years  to  prevent  cattle  and  sheep 
from  grazing  there.  Burning  of  the  cadavers  is  also  strongly  recom- 
mended, but  facilities  for  this  are  rarely  to  be  found  at  the  place  of 
death,  and  transportation  to  a  distant  place,  particularly  if  the  animal  is 
skinned,  involves  a  probable  spreading  of  the  infection.  In  Germany  the 
law  prohibits  the  skinning  of  animals  sick  with  or  dead  from  anthrax. 
If,  for  economic  reasons,  the  hides  are  to  be  saved,  they  and  the  knives 
used  in  the  skinning  must  be  properly  disinfected;  the  latter  by  boil- 
ing in  water,  the  former  by  immersion  for  a  number  of  hours  (not 
less  than  twelve)  in  strong  carbolic  acid,  creosote,  lysol,  or  other 
similar  solution.  The  process  of  tanning  does  not  kill  anthrax 
spores. 

Barns,  stalls  and  utensils  which  may  have  been  contaminated  from 
anthrax-infected  animals  must  likewise  be  disinfected; 

Vaccines  and  Serumtherapy. — Vaccination  for  the  protection  of 
animals  against  anthrax  has  been  employed  for  a  considerable  time. 
The  vaccine  used  contains  live  but  attenuated  and  sporeless  anthrax 
bacilli.  There  are  various  methods  of  decreasing  the  virulency  of 
anthrax  bacilli,  such  as  cultivation  in  the  presence  of  small  amounts 
of  antiseptics  (carbolic  acid,  bichromate  of  potash,  sulphuric  acid), 
cultivation  in  the  blood  serum  of  immune  or  immunized  animals, 
cultivation  under  higher  atmospheric  pressure,  and  cultivation  in 
successive  generations  at  comparatively  high  temperatures.  The 
latter  method  is  generally  employed  in  practice  to  obtain  vaccines  for 
the  protective  inoculation  of  domestic  animals.  The  higher  the 
temperature  under  which  the  anthrax  bacillus  has  been  grown  and 
the  longer  the  cultivation  has  been  continued  the  more  attenuated 
the  organism  becomes;  but  in  order  to  get  a  truly  protective  vaccine 
the  attenuation  must  not  be  carried  too  far.  The  method  most 
commonly  employed  is  that  of  Pasteur,  and  the  vaccines  used  are 
prepared  as  follows: 


ANTI-ANTHRAX  SERUM  251 

Vaccine  No.  1  (the  more  attenuated,  weaker  vaccine)  is  a  bouillon 
culture  of  anthrax  bacilli  grown  in  successive  generations  in  the 
incubator  at  a  temperature  of  42°  C.  for  twenty-four  days. 

Vaccine  No.  2  (the  stronger,  less  attenuated  vaccine)  is  a  bouillon 
culture  of  the  anthrax  bacillus  grown  in  the  incubator  at  the  same 
temperature  (42°  C.),  but  only  for  twelve  to  fourteen  days. 

Vaccine  No.  1,  in  small,  proper  doses,  should  kill  mice,  but  not 
guinea-pigs  or  rabbits.  Vaccine  No.  2  in  the  same  doses  should  kill 
mice  and  guinea-pigs,  but  not  rabbits.  Non-attenuated  cultures  in  the 
same  doses  kill  mice,  guinea-pigs,  and  rabbits. 

To  protect  domestic  animals  against  a  subsequent  anthrax  infection 
they  are  first  inoculated  with  the  weaker  vaccine,  No.  1,  and  after  an 
interval  of  twelve  to  fourteen  days  with  the  stronger  vaccine,  No.  2. 
The  dose  of  both  No.  1  and  No.  2  is  f  c.c.  for  horses,  mules,  and  cattle, 
and  -j-  c.c.  for  sheep  and  goats.  The  vaccines  are  injected  sub- 
cutaneously,  behind  the  shoulder  in  cattle,  inside  the  thigh  in  sheep 
and  goats,  and  on  the  side  of  the  neck  in  horses.  It  is  best  to  inject 
No.  1  on  one  side  and  No.  2  on  the  other  side. 

Vaccination  of  a  large  number  of  animals  is  attended  with  some  loss. 
Even  with  every  precaution,  and  with  carefully  prepared  vaccines  used 
properly,  the  losses  are  about  one  animal  in  several  hundred.  With 
improperly  prepared,  insufficiently  attenuated  cultures  heavy  losses 
have  occurred.  Every  fatal  case  may  become  the  source  of  spreading 
a  virulent  infection.  The  dispenser  and  user  of  the  anthrax  vaccine 
must  remember  that  cadavers  of  animals  dead  of  anthrax  vaccination 
must  be  disposed  of  in  the  same  manner  as  the  cadavers  of  animals 
dead  of  the  natural  infection  and  that  barns  and  environments  must 
be  carefully  disinfected. 

According  to  the  statistics  compiled  by  Hutyra,  the  following 
vaccinations  according  to  Pasteur's  method  were  made  in  1889-1890 
in  Hungary,  with  the  following  results: 

Head  of  horses  vaccinated 39,506 

Percentage  dead  of  anthrax  between  first  and  second  vaccination     .  0.1 
Percentage  dead  of  anthrax  during  first  year  after  second  vaccina- 
tion         0.09 

Head  of  cattle  vaccinated                .      .      ;- 718,266 

Percentage  dead  of  anthrax  between  first  and  second  vaccination       .  0.02 
Percentage  dead  of  anthrax    during  first  year  after  second  vacci- 
nation      0.02 

Head  of  sheep  vaccinated  . 1,247,231 

Percentage  dead  of  anthrax  between  first  and  second  vaccination       .  0 . 26 
Percentage   dead  of  anthrax  during  first  year  after  second  vacci- 
nation      0.33 

Anti-anthrax  Serum. — That  the  serum  of  animals  actively  immunized 
against  anthrax  possesses  specific  immune  bodies  has  been  shown  by 
Sclavo,  Marchoux,  and  Sobernheim.  These  investigators  have  pre- 
pared an  antiserum  which  they  have  used  for  prophylactic  and  thera- 
peutic purposes.  The  antiserum  is  prepared  by  hyperimmunizing 


252  ANTHRAX  BACILLUS 

such  animals  as  sheep,  horses,  and  donkeys.  These  animals  first 
receive  vaccine  No.  1  and  No.  2  in  the  usual  doses  and  intervals. 
About  two  weeks  after  the  last  vaccination  they  receive  an  injection 
of  a  more  virulent  culture.  This  injection  is  repeated  in  from  ten  to 
fourteen  days,  always  with  cultures  of  greater  virulence,  or  with 
increasing  doses  of  a  virulent  culture.  All  injections  are  made 
subcutaneously,  not  intravenously.  After  two  or  three  months  of 
this  treatment  the  serum  of  the  hyperimmunized  animals  has  obtained 
a  high  value.  If  injected  in  doses  of  20  to  25  c.c.  into  horses,  cattle, 
and  sheep  it  produces  a  passive  immunity  which  will  protect  the 
treated  animal  against  anthrax  infection  for  several  weeks,  or  perhaps 
at  best  for  two  or  three  months.  The  serum  has  also  proved  effective 
as  a  curative  agent  after  anthrax  infection  has  taken  place,  but  it 
must  then  be  used  in  larger  doses  of  30  to  150  c.c.  In  cases  of  human 
local  infections  (carbuncle)  the  antiserum  has  been  used  in  doses 
of  30  to  40  c.c.  injected  subcutaneously  in  three  or  four  different 
places  on  the  body;  in  very  bad  cases  another  set  of  injections  of  the 
same  doses  should  be  made  after  twenty-four  hours.  It  has  also 
been  used  in  bad  cases  in  10  c.c.  doses  as  an  intravenous  injection. 
Sobernheim  has  worked  out  and  used  on  animals,  with  excellent 
results,  a  combined,  simultaneous  method  of  active  and  passive 
immunization.  He  uses  vaccine  No.  2  (J  c.c.  for  horses  and  cattle, 
J  c.c.  for  sheep  and  goats),  and  injects  it  subcutaneously  into  one 
side  of  the  animal,  while  the  other  side  receives  4  to  5  c.c.  of  the 
antiserum.  The  advantages  of  this  combined  method  are  that  it 
requires  only  one  treatment  and  that  the  animals  are  fully  protected 
after  ten  to  twelve  days.  It  appears  that  the  simultaneous  method 
is  absolutely  safe  and  that  it  has  never  led  to  any  fatalities  following 
its  proper  use.  The  last  50,000  vaccinations,  according  to  Sobern- 
heim, were  made  without  a  single  death. 

It  is  well  to  state,  however,  that  nothing  is  known  at  present  con- 
cerning the  mechanism  of  the  action  of  the  antiserum.  It  does  not 
contain  an  antitoxin,  because  the  anthrax  bacillus  does  not  form 
soluble  toxins.  It  does  not  increase  phagocytosis  and  it  does  not  act 
as  a  bacteriolytic  agency.  It  acts  in  some  way  as  a  bactericidal 
antiserum.  It  seems  to  prevent  the  anthrax  bacilli  from  leaving  the 
place  of  injection  and  entering  into  and  multiplying  in  the  general 
circulation.  It  causes  them  to  die  out  in  the  subcutaneous  tissue 
after  a  comparatively  short  time,  though  by  no  means  immediately. 


QUESTIONS  253 


QUESTIONS. 

1.  What  other  names  have  been  given  to  the  disease  anthrax?    How  long 
has  it  been  known  to  mankind  ? 

2.  What  animals  are  susceptible  to  it? 

3.  Describe  the  pathologic  changes  of  anthrax. 

4.  What  is  meant  by  the  parenchymatous  degeneration  of  an  organ? 

5.  What  is  a  leukocytosis  or  hyperleukocytosis? 

6.  What  is  meant  by  a  fulminant  case  of  anthrax  or  of  an  infectious  disease 
in  general? 

7.  Describe  the  anthrax  bacillus  (a)  in  the  blood  of  an  animal  dead  from 
the  disease;  (6)  from  a  pure  culture. 

8.  Describe  some  methods  of  staining  (a)  the  capsule;  (6)  the  spores  of  the 
Bacillus  anthracis. 

9.  Describe  the  changes  in  the  bacillus  in  sporulation. 

10.  Describe  the  changes  in  the  spore  in  germination. 

11.  What  is  an  asporogenous  anthrax  bacillus? 

12.  Describe  a  gelatin  stick  culture  of  the  anthrax  bacillus. 

13.  What  is  an  impression  or  Klatsch  preparation  of  an  anthrax  colony?    How 
does  it  look? 

14.  Describe  the  growth  of  the  anthrax  bacillus  in  bouillon,  on  potatoes,  in 
milk. 

15.  Discuss  the  resistance  of  the  anthrax  bacillus  and  its  spores. 

16.  What  bacterium  is  very  antagonistic  to  the  anthrax  bacillus? 

17.  How  do  cattle  and  sheep  generally  contract  anthrax? 

18.  How  do  men  contract  the  disease? 

19.  Describe  the  steps  to  establish  beyond  doubt  the  diagnosis  of  anthrax. 

20.  How  is  anthrax-suspected  material  prepared  for  forwarding  to  a  distant 
laboratory  for  diagnosis? 

21.  How  should  anthrax  cadavers  be  disposed  of? 

22.  Describe  the  method  of  Pasteur  used  in  the  preparation  of  the  anthrax 
vaccines. 

23.  Describe  and  discuss  their  use  in  the  protection  of  cattle  and  sheep. 

24.  How  is  the  anti-anthrax  serum  prepared? 

25.  What  is  the  simultaneous  method  to  protect  animals  against  anthrax? 
What  are  its  advantages? 

26.  Describe  the  different  toxins  of  anthrax  and  their  action. . 


CHAPTEK    XXI. 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX. 

Occurrence  and  Historical. — The  disease  known  as  symptomatic 
anthrax,  black-leg,  black-quarter,  quarter-ill,  Sarcophysema  hsemor- 
rhagicum  bovis;  "Kalter  Brand,  Rauschbrand"  (German);  charbon 
symptomatique,  charbon  bacterie  (French),  generally  affects  cattle 
and  occasionally  other  animals,  like  sheep  and  hogs.  It  is  due  to  a 
specific  anaerobic  gas-forming  microorganism  known  as  the  Bacillus 
or  Clostridium  sarcophysematos  bovis,  bacillus  of  symptomatic 
anthrax  or  Bacillus  chauveaui.  The  disease  is  particularly  prevalent 
in  mountainous  countries,  with  deep  and  marshy  valleys,  also  in  flat 
countries,  in  which  the  pastures  are  exposed  to  periodical  inundations. 
It  is  most  prevalent  during  the  hot  season,  and  attacks  particularly 
cattle  between  six  months  and  two  years  old.  The  disease  has  un- 
doubtedly been  known  for  a  long  time,  but  it  was  formerly  generally 
mistaken  for  anthrax.  Bellinger  (1875)  and  Feser  (1876)  first  pointed 
out  the  difference  between  true  anthrax  and  black-leg.  They 
showed  the  specific  bacilli  in  the  emphysematous  swellings  and 
inoculated  material  containing  them  into  ruminants  and  rabbits. 
Several  French  authors  studied  the  organism  in  the  following  years, 
and  Roux  (1887)  and  Kitasato  (1889)  were  the  first  to  cultivate  it 
artificially.  The  pathology  and  bacteriology  of  the  disease  were  first 
more  extensively  investigated  by  Kitt,  whose  studies  were  made  in 
the  Bavarian  Alps.  The  disease  is  quite  prevalent  in  the  United 
States,  the  majority  of  cases  having  been  reported  from  Texas,  Okla- 
homa/Kansas,  Nebraska,  Colorado,  Indian  Territory,  and  a  number 
of  Northwestern  and  Western  States.  According  to  Moore,  infected 
localities  have  also  been  recently  found  in  New  York.  Black-leg 
occurs  in  Germany,  Austria-Hungary,  Switzerland,  and  the  other 
European  countries.  It  has  also  been  observed  in  Africa  from 
Algiers  to  the  Transvaal. 

Pathologic  Lesions. — The  most  important  objective  changes  char- 
acterizing the  disease  are  rapidly  forming,  rather  diffuse,  ill-defined 
swel  ings  of  the  skin  and  superficial  muscles,  which  are  first  very 
firm  and  solid,  but  soon  become  infiltrated  with  air  (emphysematous), 
so  that  they  are  crepitant  to  the  touch  and  tympanitic  upon  percussion. 
To  the  palpitating  finger  these  swellings  impart  a  feeling  as  if  there 
was  paper  beneath  the  skin.  The  most  common  seat  of  these 
emphysematous  lesions  is  in  the  thick  muscles  of  the  hind-  or  fore- 
legs, from  which  fact  the  names  black-leg  and  quarter-ill  are  derived. 


MORPHOLOGY  AND  STAINING  PROPERTIES  255 

The  disease  generally  takes  an  acute,  and,  as  a  rule,  fatal  course. 
On  postmortem  examination  the  cadaver  is  generally  much  distended, 
not  merely  at  the  abdomen  but  over  the  entire  external  surface 
which  is  filled  with  gas.  The  formation  of  the  latter  continues  after 
death.  The  subcutaneous  tissue  upon  removal  of  the  skin  presents 
a  gelatinous,  yellowish  or  hemorrhagic  appearance;  the  muscles 
appear  dark,  red  brown  or  black  brown.  The  soft,  necrotic  muscles 
crepitate  and  discharge  gas  on  section.  They  are  sometimes  so  full 
of  air  that  they  float  on  water  like  a  piece  of  lung.  The  meat  of 
animals  dead  from  black-leg  has  a  peculiar  sweetish,  rancid,  non- 
fetid  smell.  Sometimes  only  one  extremity  shows  these  characteristic 
changes,  at  other  times  two,  three,  or  all  four  are  involved.  While 
the  parts  affected  by  the  disease  always  contain  an  enormous  quantity 
of  blood,  the  unaffected  parts  may  be  very  pale  and  anemic.1  In 
addition  to  these  changes  hemor- 
rhages into  the  serous  membranes  FIG.  135 
and  an  accumulation  of  hemor- 
rhagic serous  fluid  in  the  various 
body  cavities  are  found.  The 
liver  and  kidneys  show  evidences 
of  parenchymatous  degeneration; 
the  former  is  sometimes  filled  with 
gas  (foamy  liver).  The  specific 
bacilli  are  found  in  the  diseased 
muscles,  serous  hemorrhagic  exu- 
dates,  and  gall-bladder,  liver,  etc. 
Many  of  the  organisms  are  gener- 
ally sporulating. 

Morphology  and  Staining  Proper- 
ties.— The     bacillus     Of      emphy-         Bacilli  of  symptomatic  anthrax,  showing 

sematous  anthrax  is  from  2  to  6  spores.    (After  Zettnow.) 

micra  long  and  0.5  to  0.8  micron 

wide.  It  generally  appears  singly,  or  in  pairs,  rarely  in  long  chains; 
some  are  cylindrical,  others  more  oval  (hence  the  name,  clostridium), 
still  others  are  club-shaped.  French  authors  have  compared  the  shape 
of  the  bacillus  to  snow-shoes.  The  clostridium  shape  is  best  seen 
after  sporulation  has  taken  place.  The  organism,  unlike  the  anthrax 
bacillus,  forms  spores  while  in  the  body  of  the  live  animal.  The 
spores  are  generally  in  the  middle  of  the  bacterium,  sometimes  more 
toward  one  end,  and  many  of  them,  particularly  in  cultures,  are 
found  free.  In  young  cultures  the  bacilli  are  quite  actively  motile 
and  possess  numerous  flagella,  which,  however,  break  off  easily  and  are 
generally  difficult  to  demonstrate  by  staining  methods.  The  bacillus 
stains  in  a  peculiar  manner  with  iodin  solution.  The  vegetative 

1  A  condition  like  this  where  an  anemia  of  one  part  is  brought  about  by  a  congestion  in  another 
part  is  called  a  collateral  anemia. 


256  BACILLUS  OF  SYMPTOMATIC  ANTHRAX 

form  then  appears  blue,  the  clostridia  black  violet,  and  the  spores 
remained  unstained.  In  the  bodies  of  sick  cattle  spore  formation  is 
generally  well  marked;  the  contrary  is  true  in  guinea-pigs  infected 
experimentally.  The  bacillus  keeps  the  stain  when  treated  by  Gram's 
method. 

Cultural  Properties. — The  black-leg  bacillus  is  a  strictly  anaerobic 
bacterium.  According  to  Kitt  the  best  method  of  obtaining  pure 
cultures  is  the  following:  With  a  very  fine  funnel,  gelatin  and  agar 
are  poured  into  glass  tubes  20  to  30  cm.  long  and  3  to  5  mm.  wide, 
which  furnishes  a  high  narrow  column  of  the  medium.  These  glass 
tubes  are  fused  at  one  end  and  closed  at  the  other  with  a  cotton  plug. 
From  four  to  ten  of  these  agar  and  gelatin  tubes  are  melted  and  cooled 
in  the  usual  manner  and  then  inoculated;  the  first  one  with  a  few 
drops  of  meat  juice  or  blood  from  a  case  of  black-leg,  the  others  in 
the  ordinary  diluting  manner,  i.  e.,  No.  2  is  inoculated  from  No.  1, 
No.  3  from  No.  2,  and  so  forth.  In  the  last  tube,  No.  10,  for  example, 
there  is  so  little  of  the  original  bacilli-containing  material  that  a  slow 
growth,  with  scanty  colony  formation,  will  occur.  Whenever  more 
abundant  material  is  used  in  inoculation  the  growth  in  the  incubator 
under  anaerobic  conditions  is  so  rapid  that  no  individual  colonies 
are  formed  and  the  culture  medium  is  broken  up  as  a  result  of  the 
extensive  gas  formation.  In  successful  attempts  with  very  high 
dilutions  delicate,  grayish,  very  finely  granular,  or  punctate  colonies 
from  0.5  to  1  mm.  in  diameter  develop.  They  sometimes  become 
larger,  more  compact,  and  less  transparent,  and  attain  the  size  of 
a  millet  seed.  The  growth  never  reaches  the  surface  of  the  medium. 
In  gelatin  the  colonies  somewhat  resemble  frog's  spawn;  they  liquefy 
the  medium  completely,  except  the  uppermost  zone,  which  is  not 
invaded  and  remains  solid.  In  the  liquefied  mass  the  growth  sinks 
to  the  bottom  and  a  whitish,  rather  scanty  cloudy  sediment  collects. 
The  addition  of  cattle  serum  to  the  gelatin  is  very  favorable  to  the 
growth.  In  nutrient  bouillon  containing  some  sterile  cattle  blood 
serum  the  growth  is  abundant,  the  medium  soon  becomes  cloudy, 
and  gas  is  formed.  A  medium  composed  of  bouillon  (10  c.c.),  to 
which  1  to  5  c.c.  of  fresh  sterile  blood  obtained  from  the  live  animal 
has  been  added,  enables  the  bacillus  to  grow  in  the  presence  of  air. 
A  good  growth  can  also  be  obtained  by  placing  a  piece  of  fresh 
muscle  (thorax  muscle  of  a  pigeon)  procured  in  a  sterile  manner  in 
the  inoculated  bouillon.  The  bacillus  also  grows  in  pure  sterile 
blood.  In  this  medium  it  forms  spores  abundantly  and  retains  its 
virulency  much  better  than  in  other  media,  in  which  it  generally 
soon  loses  its  pathogenic  properties. 

Animals  Susceptible. — The  bacillus  is  pathogenic  to  guinea-pigs, 
very  slightly  to  rabbits,  which,  on  the  contrary,  are  very  susceptible 
to  the  bacillus  of  malignant  edema.  In  natural  infection  of  cattle 
the  bacilli  gain  entrance  through  wounds  of  the  skin  into  which  mud 
from  marshy  grounds  has  penetrated.  This  was  demonstrated  long 


VACCINE  THERAPY  AND  PROTECTIVE  INOCULATION     257 

ago  by  Feser,  who  found  bacilli  like  the  black-leg  bacillus  in  such 
mud  and  succeeded  in  producing  the  disease  by  inoculation  into 
cattle  and  sheep.  Cases  of  black-leg  have  been  seen  following  cas- 
tration. It  is  also  held  that  the  disease  may  be  contracted  by  infected 
water  or  feed.  Marek  saw  a  case  in  a  hog  in  which  the  infection  had 
occurred  through  the  tonsils.  The  black-leg  bacilli  appear  to  live 
and  multiply  as  saprophytes  in  the  soil.  Direct  transmission  from 
one  animal  to  another  does  not  appear  to  occur.  In  infection  through 
the  intestines  it  is  believed  that  spores  enter  the  lymph  and  blood 
currents  and  are  carried  by  them  to  some  place  in  a  muscle  or  in 
loose  connective  tissue,  where  aerobic  bacteria  have  gained  entrance 
through  a  wound.  With  these  the  black-leg  bacillus,  or  its  spores, 
enters  into  a  symbiotic  community,  and  the  organism  is  now  enabled 
to  multiply  and  produce  the  disease. 

Resistance. — The  spores  of  the  Bacillus  sarcophysematos  bovis 
are  highly  resistant.  Sporulating  bacilli  contained  in  pieces  of  dry 
meat  remain  virulent  for  many  years  (10).  The  spores  resist  putre- 
factive processes  for  months,  and  they  are  believed  to  remain  alive  for 
a  long  time  in  manure.  Spores  contained  in  dried  pulverized  meat 
have  been  exposed  in  the  steam  sterilizer  for  five  to  six  hours  to  a 
temperature  of  100°  C.,  and  have  still  been  found  very  virulent. 
Sheep  have  been  killed  by  inoculating  them  with  0.1  to  0.2  gram  of 
material  treated  in  this  manner.  It  requires  fully  seven  hours  in 
streaming  steam  of  100°  C.  to  kill  the  spores  in  dried  meat;  dry  heat 
of  104°  C.  has  no  effect  after  seven  hours'  action.  Virulent  spores 
have  a  negative  chemotactic  effect  upon  phagocytes,  but  avirulent 
spores  freed  of  their  toxins  are  taken  up  by  phagocytes  and  destroyed. 
If  toxin-free  spores,  however,  are  protected  against  phagocytosis  by 
mechanical  means  (sand)  or  by  chemical  means  (injection  of  lactic 
acid)  they  can  still  germinate  and  produce  a  fatal  infection.  Direct 
sunlight  kills  moist  spores  in  eighteen  hours;  dried  spores  during  the 
greatest  summer  heat  in  twenty-four  hours.  Corrosive  sublimate 
has  a  comparatively  strong  disinfective  power  toward  the  spores; 
1  to  500  solution  kills  spores  from  a  culture  in  ten  minutes;  in  moist 
fresh  meat,  in  thirty  minutes;  in  dried  meat,  after  sixty  minutes' 
exposure.  The  effect  of  carbolic  acid  is  not  very  rapid;  according  to 
Kitasato,  a  5  per  cent,  solution  killed  spores  only  after  ten  hours. 
The  microorganism  is  a  saprophytic  soil  bacterium,  and  its  strong 
resistance  makes  the  disinfection  of  infected  marshy  pastures  prac- 
tically impossible;  they  either  must  be  abandoned  or  the  animals 
having  access  to  them  must  be  protected  by  inoculation. 

Vaccine  Therapy  and  Protective  Inoculation. — The  first  successful 
experiments  in  protective  methods  against  emphysematous  anthrax 
were  undertaken  by  Arloing,  Cornevin,  and  Thomas  during  the  years 
1880  to  1887.  The  first  method  used  by  French  investigators  consisted 
in  the  intravenous  injection  of  1  to  6  c.c.  of  expressed  juice  from 
infected  muscles.  This  procedure  confers  immunity,  but  is  very 
17 


258  BACILLUS  OF  SYMPTOMATIC  ANTHRAX 

dangerous  because  of  the  unavoidable  risk  of  injecting  a  portion  of 
the  juice  into  the  connective  tissue,  in  which  case  a  violent  local 
infection  generally  leading  to  death  results.  Even  when  all  the  juice 
is  injected  into  the  veins,  there  still  remains  the  danger  of  a  fatal 
local  infection  at  some  point  where  an  accidental  hemorrhagic  con- 
tusion in  the  subcutaneous  or  muscular  tissue  has  occurred.  This 
fact  has  given  rise  to  the  belief  that  emphysematous  anthrax  is  often 
an  intestinal  infection,  as  already  stated.  Thomas'  vaccination 
method,  known  as  the  "vaccination  par  le  fil  virulent,"  was  the  first  to 
be  practised  more  extensively.  The  procedure  consists  in  saturating 
a  bundle  of  silk  threads  with  an  attenuated  culture  of  black-leg 
bacilli  which  have  passed  through  the  body  of  the  frog.  The  silk 
threads  are  inserted  in  the  tail  by  a  special  vaccination  needle 
devised  by  Thomas.  This  method  has  been  used  in  France  on 
at  least  a  million  and  a  half  head  of  cattle,  with  good  results. 
Later,  the  French  workers  prepared  two  vaccines,  one  considerably 
attenuated,  which  was  used  for  the  first  inoculation  and  one  less 
attenuated  for  the  second.  Kitt's  experiments  on  the  resistance  of 
the  black-leg  bacillus  and  its  spores  led  him  to  work  out  what  is  now 
the  most  commonly  used  method  in  the  protective  inoculation  of 
cattle  against  natural  black-leg  infection.  Dried  and  ground-up 
muscles  from  cattle  dead  from  emphysematous  anthrax  are  exposed 
for  five  or  six  hours  to  steam  of  a  temperature  of  97°  C.,  after  which 
the  powder  is  again  completely  dried.  It  was  found  that  this  powder 
in  doses  of  0.2  to  0.6  gram  still  killed  sheep,  but  in  smaller  doses 
immunized  and  protected  them.  Kitt's  method,  used  with  excellent 
results  in  Europe,  was  somewhat  modified  in  the  United  States  by 
Noergaard,  of  the  Bureau  of  Animal  Industry,  who  exposed  the 
ground-up  infected  muscles  from  cattle  for  six  hours  to  93°  to  95°  C. 
The  latter  vaccine  has  been  successfully  used  on  several  million  head 
of  cattle.  It  may  be  remembered,  however,  that  occasionally  an 
animal  treated  with  the  vaccine  dies  from  the  inoculation,  but  this 
number  is  so  small  as  to  become  a  negligible  quantity  when  com- 
pared with  the  great  protective  value  of  the  vaccination. 

Directions  for  the  Use  of  Black-leg  Vaccine. — The  following  is  an 
abstract  of  the  directions  given  by  tbe  Bureau  of  Animal  Industry  for 
the  use  of  its  prepared  vaccine:  The  black-leg  vaccine,  as  prepared 
by  the  Bureau,  consists  of  a  brownish  powder,  which  is  put  up  in 
packets  containing  either  ten  or  twenty-five  doses  each.  To  prepare 
this  powder  in  such  a  way  that  it  may  be  injected  hypodermically, 
it  is  necessary  to  use  certain  implements.  The  outfit  consists  of  a 
porcelain  mortar,  with  pestle,  a  small  glass  funnel,  a  measuring  glass, 
and  a  syringe.  For  filtering  the  vaccine,  absorbent  cotton  is  most 
suitable.  The  syringe  used  has  a  capacity  of  5  cubic  centimeters, 
and  the  piston  is  graduated  from  one  to  five,  eaeh  division  being 
subdivided  with  half  and  quarter  notches.  The  screw-regulator  may 
be  placed  at  any  mark  on  the  piston,  thus  insuring  that  the  animal 


DIRECTIONS  FOR  THE  USE  OF  BLACK-LEG  VACCINE     259 

to  be  vaccinated  receives  only  the  exact  dose  intended  for  it.  The 
plunger  is  made  of  rubber;  it  should  be  air-tight  in  the  glass  barrel, 
and  yet  capable  of  being  moved  up  and  down  smoothly.  In  order 
to  prevent  the  plungers  and  washers  from  drying  out,  the  small  loose 
cap  should  be  always  tightly  adjusted  to  the  peg  when  the  syringe 
is  not  in  use.  The  hypodermic  needles  should  be  kept  very  sharp  at 
the  point,  in  order  to  pass  easily  through  the  skin,  and  when  not  in 
use  should  have  a  fine  brass  wire  passed  through  each  to  prevent 
rusting  on  the  inside.  Whenever  the  point  of  the  needle  gets  blunt 
it  becomes  very  difficult  to  pass  it  through  the  skin,  causing  the  fingers 
of  the  operator  to  become  sore  from  attempting  to  force  it  through, 
and  frequently  the  needle  either  bends  or  breaks.  It  is,  therefore, 
of  importance  to  have  a  small  oilstone  at  hand  on  which  to  sharpen 
the  point  of  the  needle. 

Before  preparing  the  vaccine  all  the  utensils,  together  with  the 
syringe,  must  be  sterilized  thoroughly.  This  is  done  by  putting  the 
mortar,  pestle,  measuring  glass,  funnel,  and  needles  in  a  pan  of  cold 
water  and  placing  them  over  the  fire.  After  boiling  for  ten  minutes 
the  pan  with  the  contents  should  be  allowed  to  cool  off  slowly;  then 
remove  the  utensils  from  the  water  and  wipe  them  dry  with  a  clean 
linen  cloth  which  has  been  previously  boiled. 

Preparation  of  the  Vaccine. — Place  the  contents  of  one  packet  of 
the  vaccine  in  a  porcelain  mortar  and  add  a  few  drops  of  boiled 
water.  Work  the  powder  thoroughly  with  the  pestle  and  then  add, 
little  by  little,  as  many  cubic  centimeters  of  water  as  the  packet 
contains  doses.  As  the  syringe  contains  exactly  5  c.c.  it  may  be 
used  for  measuring  the  water.  Place  a  small  piece  of  absorbent 
cotton  in  the  funnel  and  press  it  lightly  into  the  upper  end  of  the 
neck,  sufficient  to  keep  it  in  place;  moisten  the  cotton  with  a  few 
drops  of  boiled  water  and  let  it  drip  off.  Stir  the  mixture  in  the 
mortar  thoroughly,  and  before  it  has  had  time  to  settle,  pour  it  into 
the  funnel  under  which  the  measuring  glass  has  been  placed.  The 
solution  should  not  be  perfectly  clear.  If  this  is  the  case,  the  cotton 
has  been  pressed  too  closely  into  the  neck  of  the  funnel. 

Age  of  Animal  and  Dosage. — Calves,  as  a  rule,  should  not  be 
vaccinated  until  they  are  six  months  old.  Under  this  age  they  are 
practically  immune,  and  it  has  been  claimed  that  when  vaccinated 
before  they  are  six  months  old  they  are  likely  to  lose  the  artificial 
immunity  induced  and  become  susceptible  again.  Animals  over  two 
years  old  are  rarely  affected,  and  the  mortality  is  so  small  as  to  make 
vaccination  unprofitable.  It  is  the  animals  between  six  months 
and  two  years  old  which  should  be  vaccinated.  Vaccination  and 
castration  should  not  be  performed  at  the  same  time. 

Ten  days  to  two  weeks  should  be  allowed  to  elapse  after  vacci- 
nation before  any  surgical  operation  is  undertaken,  and  if  performed 
before  the  vaccination,  ample  time  should  be  allowed  for  the  part 
to  heal  and  the  animal  to  regain  its  lost  strength.  Animals  one  year 


260  BACILLUS  OF  SYMPTOMATIC  ANTHRAX 

old  or  over  are  injected  with  a  full  dose  of  vaccine;  that  is,  1  c.c.  of 
the  solution.  Under  this  age  the  dose  may  be  reduced  to  one-half 
or  three-fourths  of  a  full  dose,  according  to  the  size  and  development 
of  the  animal.  Less  than  one-half  dose  should  never  be  injected. 
In  determining  the  dose  for  each  animal,  more  consideration  should 
be  given  to  the  size  and.  development  of  the  animal  than  to  its  exact 
age.  The  most  convenient  place  for  inoculation  is  on  the  side  of  the 
neck,  just  in  front  of  the  shoulder,  where  the  skin  is  loose  and  rather 
thin.  If  the  animals  are  secured  in  a  dehorning  chute,  it  is  easier  to 
vaccinate  them  on  the  side  of  the  chest  just  behind  the  shoulder. 
Steps  in  the  Vaccination  Process. — 1.  Sterilize  outfit  by  boiling. 

2.  Place  the  contents  of  one  packet  in  the  mortar  and  add  a  few 
drops  of  water. 

3.  Work  the  mixture  well  with  the  pestle. 

4.  Add  two  to  five  syringefuls  of  water,  according  to  the  size  of 
the  packet,  and  stir  well. 

5.  Place  cotton  in  glass  funnel  and  moisten  with  water. 

6.  Filter  vaccine  into  the  glass  or  bottle. 

7.  Secure  the  animal  to  be  injected. 

8.  Insert  the  needle  through  the  skin. 

9.  Fill  the  syringe  and  adjust  the  screw  regulator  on  the  piston. 
If  the  first  animal  is  a  yearling  or  older,  place  regulator  No.  1  on  the 
syringe. 

10.  Fit  the  peg  of  the  syringe  into  the  cap  of  the  needle  and  inject 
the  dose. 

11.  Withdraw  syringe   and   needle   together.     If  the   syringe   is 
removed  from  the  needle  before  the  latter  has  been  drawn  out  of  the 
skin  some  of  the  injected  vaccine  will  flow  back  through  the  needle 
and  be  lost.    In  this  case  the  animal  does  not  get  a  full  dose,  and  will 
consequently  be  insufficiently  protected. 

Black-leg  Toxins  and  Antitoxins. — If  cultures  of  the  bacillus  of 
emphysematous  anthrax  in  fluid  culture  media  are  filtered  through 
a  Pasteur-Chamberland  or  Berkefeld  filter,  soluble  toxins  cannot  be 
demonstrated  in  the  filtrate.  Grassberger  and  Schattenfroh,  however, 
have  devised  a  somewhat  complicated  method  of  growing  the  organism 
in  particular  media  and  filtering  its  cultures  through  powdered 
chalk  in  such  a  manner  that  a  germ-free  filtrate  containing  a 
powerful  toxin  is  obtained.  The  properties  of  the  latter  are 
described  as  follows:  The  toxin  seems  to  be  pathogenic  for  all 
warm-blooded  animals;  its  effect  seems  to  be  the  same,  calculated 
per  body  weight  of  animal  in  the  case  of  cattle,  sheep,  guinea-pigs, 
rabbits,  monkeys,  dogs,  hedgehogs,  mice,  chickens,  and  pigeons. 
Cold-blooded  animals  are  not  susceptible.  After  injection  of  a 
single  fatal  dose  into  a  guinea-pig,  a  painful,  doughy  or  elastic 
swelling,  which  spreads  after  eight  to  ten  hours,  is  formed  at  the 
place  of  injection.  The  skin  early  shows  hemorrhages.  There  is  first 
a  slight  elevation  of  temperature,  which  later  becomes  subnormal. 


QUESTIONS  261 

The  animal  becomes  restless  and  dies  within  two  to  four  days,  a 
hemorrhagic  discharge  from  the  mouth  arid  nostrils  appearing  shortly 
before  death.  The  postmortem  examination  shows  a  hemorrhagic 
oedema  of  the  subcutaneous  tissue  and  heniorrhagic  serous  exudates 
in  the  body  cavities.  The  two  authors  named  have  succeeded  in 
obtaining  germ-free  solutions,  of  which  two  milligrams  or  even  one- 
half  milligram  would  kill  a  guinea-pig  of  one-half  pound  weight 
(250  grams).  The  toxin  which  has  these  pathogenic  properties  is 
not  very  stable;  it  is  destroyed  in  one  hour  if  heated  to  50°  C.,  1  per 
cent,  carbolic  acid  solution  destroys  it  in  twenty-four  hours,  but 
chloroform  has  no  deleterious  effect  upon  it,  and  is  used  as  a  means 
for  its  conservation.  The  authors  also  succeeded  in  preparing  an 
antitoxin.  Guinea-pigs  could  not  be  actively  immunized  against  the 
toxin,  on  the  contrary,  a  repeated  injection  of  small  doses  produced 
a  hypersusceptibility.  Rabbits,  however,  can  be  actively  immunized, 
and  also  cattle.  The  blood  serum  of  cattle  after  a  systematic  course 
of  hyperimmunization  lasting  four  to  five  months  often  develops  a 
high  antitoxic  or  immunizing  value,  and  the  antitoxin  has  the  great 
advantage  of  being  apparently  quite  stable  when  properly  kept  and 
stored.  It  was  found  to  be  thermostabile,  and  heating  it  for  three 
hours  at  60°  C.  had  no  detrimental  effect  upon  it.  The  "Rauschbrand" 
antitoxin  of  Grassberger  and  Schattenfroh,  however,  is  valueless  in 
the  treatment  of  natural  or  artificial  infection  with  the  black-leg 
bacillus,  because  death  is  not  caused  by  the  soluble  toxin  but  by 
factors  which  are  in  no  way  affected  by  the  antitoxin.  The  latter, 
particularly  on  account  of  its  very  peculiar  behavior  toward  its  toxin, 
is,  therefore,  of  theoretical  interest  only. 

QUESTIONS. 

1.  What  are  the  other  names  of  the  disease  black-leg? 

2.  What  is  the  cause  of  this  disease  of  cattle? 

3.  At  what  age  are  cattle  most  susceptible  to  the  disease? 

4.  Describe  the  most  characteristic  pathologic  lesions  of  symptomatic  anthrax. 

5.  What  is  the  meaning  of  the  terms  emphysematous,  crepitant,  tympanitic? 

6.  What  is  a  collateral  anemia?    Explain  its  occurrence  in  black-leg. 

7.  Describe  the  morphology  and  staining  properties  of   the  Bacillus  sarco- 
physematos  bovis. 

8.  What  is  a  clostridium? 

9.  Describe  the  cultural  properties  of  the  bacillus  of  black-leg. 

10.  What  substance  added  to  the  ordinary  culture  media  greatly  favors  the 
development  of  the  organism? 

11.  Describe  Kitt's  method  of  cultivating  the  bacillus  from  the  juice  of  infected 
meat  or  from  a  small  piece  of  the  latter. 

12.  Describe  the  differences  in  the  action  of  the  anthrax  bacillus  and  the 
Bacillus  sarcophysematos  bovis  in  the  blood  of  animals  dying  from  these  two 
diseases,  respectively. 

13.  What  animal  may  be  used  to  differentiate  by  inoculation  experiments 
between  the  black-leg  bacillus  and  that  of  malignant  edema? 

14.  What  is  the  natural  mode  of  infection  of  symptomatic  anthrax  in  cattle? 

15.  Discuss  the  resistance  of  the  spores  of  the  bacillus. 

16.  Which  spores  are  and  which  are  not  destroyed  by  phagocytosis?     How 
can  the  phagocytosis  of  toxin  free  spores  be  prevented? 


262  BACILLUS  OF  SYMPTOMATIC  ANTHRAX 

17.  How  can  pastures  infected  with  the  black-leg  bacillus  and  its  spores  be 
disinfected? 

18.  Describe  the  first  experiments  in  protective  inoculation  against  "Rausch- 
brand"  infection. 

19.  What  are  the  dangers  of  the  first  method  employed  by  Arloing,  Cornevin, 
and  Thomas? 

20.  What  is  Thomas'  method  of  vaccination? 

21.  What  is  Kitt's  method?     What  is  the   method  generally  employed  iii 
the  United  States? 

22.  Describe  in  detail  the  steps  in  the  use  of  black-leg  vaccine  prepared  by  the 
Bureau  of  Animal  Industry. 

23.  What  kind  of  animals  should  be  inoculated;  what  kind  should  not? 

24.  Does  the  bacillus  of  symptomatic  anthrax  form  a  soluble  toxin?  how  can 
it  be  obtained?  what  are  its  properties? 

25.  Can  an  immune  serum  against  such  a  toxin  be  prepared?    What  are  its 
effects  in  the  prevention  and  cure  of  the  natural  disease? 


CHAPTER    XXII. 

THE  BACILLUS  OF  MALIGNANT   EDEMA  AND  SIMILAR  BACTERIA 

—BACILLUS  OF  GASTROMYCOSIS  OVIS— BACILLUS 

AEROGENES  CAPSULATUS. 

BACILLUS  OF  MALIGNANT  EDEMA. 

Occurrence  and  Historical. — A  spore-forming  anaerobic  bacterium, 
causing  a  peculiar  form  of  wound  infection  and  inflammation,  fre- 
quently occurs  in  soil,  sewage,  dust,  grasses,  the  intestinal  contents  of 
animals,  manure,  and  putrefying  animal  substances.  Feser,  in  1876, 
working  with  "Rauschbrand,"  evidently  saw  these  bacilli,  but  they 
were  first  more  exactly  recognized  by  Pasteur,  who  was  able  to 
produce  in  guinea-pigs  and  rabbits  by  the  inoculation  of  putrid 
material  a  disease  characterized  by  an  edematous  inflammation  at  the 
place  of  inoculation.  Pasteur  named  the  bacterium  which  produced 
these  changes  "Vibrion  septique."  Robert  Koch,  in  1881,  showed 
that  this  disease  was  not  a  true  septicemia,  and  he  called  it  malignant 
edema  and  the  microorganism  the  bacillus  of  malignant  edema.  Its 
pathogenic  significance  was  later  studied  by  a  number  of  observers, 
including  Brieger  and  Ehrlich,  Jensen  and  Sand,  Kitt,  and  others. 
It  has  been  found  to  be  the  cause  of  wound  complications  in  man  and 
domestic  animals,  among  the  latter  particularly  in  horses,  cattle,  and 
sheep,  very  rarely  in  hogs,  dogs,  and  cats. 

Pathologic  Lesions. — At  the  place  of  infection  with  the  bacillus  of 
malignant  edema  the  tissues  are  swollen  and  considerably  infiltrated 
with  a  yellowish  or  reddish  serous  exudate.  The  fluid  contains 
numerous  gas-bubbles.  Hemorrhages  are  found  here  and  there  in 
the  tissues.  The  peritoneal  cavity  contains  a  moderate  amount  of 
reddish  serous  fluid;  the  peritoneum  is  congested,  dull,  but  does 
not  show  any  fibrinous  deposit.  In  cases  where  malignant  edema 
follows  delivery  the  uterus  is  found  to  be  flaccid  and  insufficiently 
contracted;  the  wall  of  the  uterus  and  the  pelvic  connective  tissue 
are  edematous.  The  cotyledons,  according  to  Carl,  are  changed 
into  a  mushy,  dirty,  ill-smelling  mass.  The  spleen,  as  a  rule,  is  not 
much  changed,  but  it  may  be  swollen,  edematous,  and  emphysema- 
tous.  The  liver  shows  parenchymatous  degeneration,  the  lymph 
nodes  are  swollen,  the  lungs  are  edematous  and  congested,  and  the 
heart  muscle  is  soft  and  flabby;  the  blood,  as  in  anthrax,  does  not 
promptly  and  firmly  coagulate,  and  putrefactive  changes  set  in  early. 
If  juice  from  an  emphysematous  organ  or  location  is  examined 


264  THE  BACILLUS  OF  MALIGNANT  EDEMA 

microscopically  long,  slender  rods  are  seen;  often  also  pseudofilaments. 
The  bacilli  somewhat  resemble  anthrax  bacilli,  but  have  rounded 
and  sometimes  even  pointed  ends,  instead  of  square  ends. 

Morphology  and   Staining  Properties. — The  bacillus   of  malignant 
edema  varies  much  in  length;  its  average  is  from  2  to  4  micra;  some 
individual  bacteria  are  much  longer,  and  the  pseudofilaments  may 
have  a  length  of  15  micra.    The  diameter  of 
FIG.  136  the  rods   and    pseudofilaments   is  generally 

^  1   micron.     Spores  are  found  in  moderate 

.  numbers  in   the   serous   exudates;  they  are 

*y  very  numerous  in  cultures.     They  are,  how- 

/    A          ever,  only  found  in  the  single  bacilli,  not  in 
/  the  filaments;  they  are  oval,  generally  situ- 

ated in  the  middle,  occasionally  toward  the 
Bacillus  of  malignant  edema,     end  of  the  rods;  they  bulge  out  the  bacillus 
(Park.)  somewhat,  but  not  to  such  an  extent  as  the 

symptomatic  anthrax  bacilli.     Jensen  found 

that  some  stems  of  malignant  edema  bacilli  form  pseudofilaments 
either  in  the  cultures  or  in  bodies  of  animals.  The  bacillus  is  motile 
and  possesses  numerous  flagella.  The  organism  stains  with  the 
ordinary  watery  anilin  stains  and  keeps  Gram's  stain  if  the  decolor- 
ization  in  alcohol  is  not  continued  very  long. 

Cultural  Properties. — The  organism  is  strictly  anaerobic.  It  grows 
at  room  temperature  and  in  the  incubator  in  the  absence  of  oxygen, 
on  all  the  ordinary  media,  particularly  well  in  the  presence  of  a  salt 
of  formic  acid  or  glucose.  In  gelatin  stick  cultures,  small,  white, 
shining  round  colonies  are  formed  along  the  stab.  As  these  increase 
in  size  gas-bubbles  appear  and  the  medium  becomes  liquefied  and  is 
changed  into  a  grayish-white  cloudy  fluid.  Agar  becomes  cleft  and 
torn  in  consequence  of  the  gas  formation  during  the  growth.  Bouillon 
becomes  cloudy  and  forms  a  whitish  sediment  after  two  to  three 
days;  the  upper  strata  then  clear  up  and  small  gas-bubbles  continue 
to  rise  to  the  surface.  The  bacillus  grows  well  on  coagulated  blood 
serum.  On  potatoes  it  multiplies  likewise,  but  the  growth  which  it 
forms  is  invisible.  When  growing  in  blood  serum  the  bacillus  produces 
a  very  fetid  smell,  due  to  the  putrid  decomposition  of  the  serum 
albumin. 

Resistance. — The  spores  of  the  bacillus  of  malignant  edema  are 
very  resistant,  and  in  this  respect  act  much  like  the  spores  of  the 
Bacillus  sarcophysematos  bovis.  Sunlight  appears  to  have  little 
effect  upon  them,  likewise  5  per  cent,  carbolic  acid. 

Natural  Infection. — This  generally  takes  place  from  cutaneous 
wounds,  from  the  denuded  surface  of  the  parturient  uterus,  etc.  In- 
fections in  man  and  the  horse  have  been  seen  following  medicinal 
injections  with  unclean  hypodermic  syringes.  The  mucous  mem- 
branes of  the  mouth  and  pharynx  also  form  a  portal  of  entrance.  It 
is  believed  that  the  organism  or  its  spores  may  enter  the  circulation 


BACILLUS  OF  GASTROMYCOSIS  OVIS  265 

from  the  intestines  and  may  be  carried  to  a  contused  place  which 
furnishes  a  favorable  location  for  multiplication.  Kitt  has  reported 
cases  in  which  it  appears  that  sheep  may  contract  a  malignant  edema 
of  the  lungs  by  inhalation. 

In  natural  infection  the  bacilli  are  found  in  the  serous  or  watery 
exudates.  They  occur  in  enormous  numbers  in  the  connective  tissue 
of  the  affected  part,  in  more  moderate  numbers  in  the  superficial 
portions  of  the  muscles  and  very  scantily  in  its  deeper  substance. 

Artificial  Inoculation. — Equines,  guinea-pigs,  rabbits,  rodents,  cattle, 
chickens,  and  pigeons  are  susceptible  to  artificial  inoculation;  dogs 
and  cats  only  very  slightly. 

Protective  Inoculation. — Leclainche  and  Valee  have  shown  that 
animals  may  be  protected  against  malignant  edema  infection  by 
inoculation  of  spore-containing  material  which  has  been  heated  for 
seven  hours  at  92°  C.  Animals  repeatedly  treated  with  such  vaccines 
also  develop  an  immune  serum  which  will  produce  passive  immunity 
in  other  animals.  All  these  tests  are  still  in  an  experimental  stage,  and 
neither  protective  virus  inoculation  nor  passive  immunization  has 
ever  been  used  in  practice. 


BACILLUS  OF  GASTROMYCOSIS  OVIS. 

Occurrence. — In  the  northern  parts  of  Europe,  Iceland,  Norway, 
the  Faroe  Islands,  Scotland,  Denmark,  and  also  in  North  Germany, 
a  disease  of  sheep  occurs  which  is  known  as  bradsot,  braasod,  brada- 
pestina,  bradasottina,  bradafarid,  braxy,  or  technically,  as  gastro- 
mycosis  ovis.  The  disease  runs  a  very  rapid  course.  A  sheep  appar- 
ently well  in  the  evening  may  be  dead  the  following  morning.  The 
disease  was  described  as  early  as  about  the  middle  of  the  eighteenth 
century.  The  first  bacteriologic  examinations  were  made  by  Kingberg, 
and  the  first  description  of  the  bacillus  of  bradsot  was  published  by 
Nielsen,  whose  findings  were  confirmed  and  extended  by  Jensen. 

Pathologic  Changes. — Jensen  describes  these  as  follows :  The  mucosa 
of  the  stomach  and  small  intestine  exhibits  a  serous  hemorrhagic 
infiltration,  likewise  the  abdominal  connective  tissue,  which  may  also 
show  infiltration  with  gas.  The  mucosa  of  the  "lab"  stomach  (rennet) 
is  sometimes  necrotic,  and  upon  microscopic  examination  the  tissues 
here  show  enormous  numbers  of  bradsot  bacilli.  When  the  disease 
is  produced  by  the  experimental  subcutaneous  inoculation  the 
animals  develop  hemorrhagic  infiltrations  of  the  deeper  muscles, 
often  with  gas  infiltration,  and  the  pathological  picture  very  much 
resembles  symptomatic  anthrax.  Jensen  failed  to  produce  bradsot 
in  sheep  by  feeding  them  with  hard  material,  including  thistles 
which  had  been  contaminated  with  cultures  of  the  bacilli. 

Morphology. — The  bacillus  of  gastromycosis  ovis  is  a  large  rod,  2 
to  6  micra  long,  1  micron  wide.  It  has  rounded  ends,  and  is  found 


266  THE  BACILLUS  OF  MALIGNANT  EDEMA 

singly  in  the  serous  cavities  of  sheep  dead  from  the  disease,  and 
frequently  in  longer  chains  and  pseudofilaments  in  the  interior  of 
the  parenchymatous  organs.  It  forms  spores  in  the  body  of  the 
infected  animal,  and  also  rapidly  in  artificial  cultures.  The  spores  are 
generally  in  the  middle,  more  rarely  toward  one  end  of  the  bacillus. 
The  organism  is  motile,  and  possesses  flagella. 

Cultural  Properties. — The  bacillus,  under  anaerobic  conditions,  grows 
in  the  ordinary  culture  media;  but  a  more  abundant  growth  develops 
only  upon  the  addition  of  a  moderate  amount  of  glucose,  which  is 
fermented  with  gas  formation.  It  grows  well  in  milk,  which  it  coagu- 
lates in  consequence  of  abundant  acid  formation.  The  coagulated 
casein  is  not  peptonized.  The  organism  does  not  grow  in  a  medium 
of  acid  reaction,  and  when  it  has,  in  its  growth  in  an  alkaline  medium, 
formed  a  certain  amount  of  acid  from  glucose  or  lactose,  the  develop- 
ment ceases.  It  is  very  probable  that  the  bacillus  occurs  in  the  upper 
strata  of  the  soil,  but  this  has  not  been  definitely  established. 


OTHER  ANAEROBIC  GAS-FORMING  BACILLI. 

Kitt  discusses  a  number  of  affections  in  animals  under  the  name 
of  "pseudo-Rauschbrand."  These  are  either  clearly  due  to  the 
bacillus  of  malignant  edema  or  one  of  its  varieties  or  organisms  more 
nearly  related  to  the  Bacillus  sarcophysematos  bovis.  According  to 
Kitt  and  to  Carl  the  disease  of  cattle  called  in  German  "Geburts- 
rauschbrand"  (parturient  emphysema)  is  due  to  a  typical  bacillus  of 
malignant  edema.  This  bacterium  has  also  been  found  by  Jensen, 
Home,  Hutyra,  and  Kitt  as  the  caue  of  the  same  disease  in  horses. 
The  Bacillus  cedematis  thermophilus,  found  by  Kerr  and  Novy  in  a 
cow,  is  probably  a  variety  of  the  Bacillus  sarcophysematos  bovis,  from 
which  it  differs  in  certain  minor  cultural  features,  and  in  the  fact  that 
it  is  very  pathogenic  for  rabbits  and  rats,  which  are  relatively  resistant 
to  the  typical  bacillus  of  symptomatic  anthrax. 

A  peculiarly  interesting  member  of  the  group  is  a  bacillus  patho- 
genic for  whales  described  by  Nielsen.  For  centuries  the  inhabitants 
of  northern  Norway  have  caught  whales  in  a  unique  manner.  They 
noticed  that  these  animals  sometimes  suffered  from  emphysematous 
inflammations  of  the  muscles,  and  they  poison  arrows  by  dipping 
them  into  the  juices  of  such  diseased  meat.  If  whales  are  hit  by  such 
poisoned  arrows  they  sicken  rapidly  (within  eighteen  to  thirty-six 
hours)  and  can  be  easily  caught.  Nielsen,  who  examined  the  emphy- 
sematous whale  meat  and  the  poisoned  arrows,  found  an  organism 
of  the  Bacillus  sarcophysematos  bovis  type  in  enormous  numbers. 
The  muscles  of  the  whale  infected  with  the  bacillus  of  Nielsen  show 
the  same  anatomical  changes  as  the  meat  of  cattle  sick  with  emphy- 
sematous anthrax. 

Reindeer  Plague. — Among  the  reindeer  of  Lapland  an  occasionally 
very  epidemic  disease  which  kills  thousands  of  calves  and  young 


BACILLUS  AEROGENES  CAPSULATUS  267 

animals  has  been  observed.  The  cadavers  show  emphysematous 
edema  over  various  parts  of  the  body.  Hemorrhages  from  the  nose 
generally  precede  the  fatal  termination.  Lungdren  and  Bergman 
found  a  bacterium  much  like  the  symptomatic  anthrax  bacillus  in  the 
edematous  fluid  and  serous  exudates  of  the  sick  and  dead  animals. 
The  bacillus  of  reindeer  plague  grows  best  aerobically;  it  is  1.6  to  4.8 
micra  long,  0.7  to  0.8  micron  wide;  motile,  with  flagella;  it  forms 
oval  spores  in  the  centre  or  at  one  end;  occurs  singly,  in  pairs  or  in 
chains  and  pseudofilaments,  and  stains  with  the  watery  anilin  stains 
and  by  Gram's  method.  It  grows  between  12°  and  38°  C.  The 
spores  are  very  resistant,  and  the  organism  is  pathogenic  in  experi- 
mental inoculation  to  reindeer,  sheep,  guinea-pigs,  white  mice, 
chickens,  pigeons,  and  sparrows. 

Other  gas-forming,  edema-producing  bacilli  which  have  been 
described,  but  which  are  not  strictly  anaerobic,  are  the  following: 

Novy's  Bacillus  cedematis  maligni  II,  Klein's  Bacillus  osdematis 
sporogenes,  and  Sanfelice's  Bacillus  cedematis  aerogenes. 

BACILLUS  AEROGENES  CAPSULATUS  (WELCH),   OR  BACILLUS 
EMPHYSEMATOSUS  (FRAENKEL). 

This  bacillus,  described  by  Welch,  Fraenkel,  Nuttall,  Flexner, 
Howard,  Jr.,  and  others,  is  an  anaerobic  gas-forming  sporogenous 
microorganism,  and  has  been  found  a  number  of  times  as  the  cause 
of  terminal  infections  in  man.  It  belongs  to  the  group  of  malignant 

FIG.  137 


Bacillus  aerogenes  cupsulatus  infecting  a  human  liver.     X  1000.   (Author's  preparation.) 

edema  and  symptomatic  anthrax  bacilli,  but  has  never  been  en- 
countered as  a  cause  of  natural  infection  in  animals.  It  has  recently 
been  found  by  MacNeal,  Latzer,  and  Kerr  as  a  normal  inhabitant 
of  the  intestines  of  healthy  persons,  and  it  is  probably  also  found  in 


268  THE  BACILLUS  OF  MALIGNANT  EDEMA 

the  soil.  The  bacillus  is  from  3  to  5  micra  long  and  about  1  micron 
in  diameter;  it  generally  occurs  in  pairs  and  irregular  groups,  rarely 
if  ever  in  chains.  It  has  rounded  ends,  is  not  motile,  possesses  no 
flagella,  stains  by  the  ordinary  methods,  and  keeps  Gram's  stain. 
It  forms  spores  but  not  in  the  infected  human  body,  only  in  artificial 
culture  media,  best  on  Loeffler's  blood-serum  mixture.  The  vegetative 
forms  of  the  organism  are  not  very  resistant;  the  spores  are,  however, 
quite  resistant.  In  experimental  work  the  bacillus  has  been  shown 
to  be  pathogenic  to  rabbits,  guinea-pigs,  and  pigeons. 


QUESTIONS. 

1.  What  is  the  bacillus  of  malignant  edema?  'Where  is  it  found? 

2.  What  relation  has  the  vibrion  septique  to  the  Bacillus  redema  maligni? 

3.  What  beings  are  susceptible  to  infection  with  this  bacillus? 

4.  Describe  its  most  characteristic  pathologic  lesions. 

5.  What  is  the  character  of  the  blood  after  death  due  to  infection  with  this 
bacterium? 

6.  Describe  the  morphology  of  the  bacillus.     In  what  morphologic  features 
does  it  differ  from  the  anthrax  and  from  the  symptomatic  anthrax  bacilli? 

7.  Describe  the  spores  of  the  malignant  edema  bacillus. 

8.  Describe  its  cultural  properties. 

9.  Discuss  its  resistance. 

10.  How  does  natural  infection  take  place? 

11.  What  animals  are  susceptible? 

12.  What  work  has  been  done  as  to  protective  inoculation  against  malignant 
edema? 

13.  What  is  bradsot,  or  gastromycosis  ovis?    Where  does  it  occur? 

14.  Describe  the  pathologic  changes  of  the  disease. 

15.  Describe  the  bradsot  bacillus. 

16.  What  is  meant  by  pseudo-Rauschbrand  bacilli? 

17.  What  is  parturient  emphysema  due  to? 

18.  What  is  the  Bacillus  cedematis  thermophilus? 

19.  Describe  the  Nielsen  gas  bacillus  of  whales. 

20.  What  is  reindeer  plague?     What  bacillus  causes  it?     Describe  its  mor- 
phology, cultural  properties,  and  pathogenesis. 

21.  Name  some  edema-producing  aerobic  gas  bacilli. 

22.  What   is  the   Bacillus   aerogenes   capsulatus?     What   human  and  what 
animal  diseases  does  it  cause? 

23.  Where  is  it  found? 


CHAPTEE    XXIII. 

BACILLUS  OF  TETANUS. 

Occurrence  and  Animals  Susceptible. — Tetanus,  or  lockjaw,  is  a 
disease  of  man  and  some  of  the  lower  animals,  which  has  been 
known  for  a  long  time.  It  is,  in  fact,  mentioned  by  Hippocrates,  and 
it  had  been  noticed  that  it  frequently  follows  lacerated,  deep-seated 
contaminated  wounds.  The  disease  is  characterized  by  clonic  and 
tonic  convulsions  of  the  voluntary  muscles,  but  it  has  no  charac- 
teristic anatomic  or  histopathologic  lesions,  and  a  diagnosis  of 
tetanus  cannot  be  made  from  a  ^postmortem  examination  unless  it 
is  combined  with  a  bacteriologic  examination,  including  animal  inoc- 
ulation experiments.  The  clinical  symptoms,  however,  are  so  charac- 
teristic that  it  is  easy  to  diagnosticate  a  case  of  typical  tetanus  in  man 
or  animals.  The  disease,  as  a  rule,  follows  wound  infection;  the  wound 
so  infected  may  be  the  umbilicus  of  the  newborn  or  the  uterus  after 
parturition.  Man  and  the  horse  are  most  susceptible  to  natural  infec- 
tion, but  cattle,  sheep,  and  hogs  are  likewise  subject  to  tetanus.  The 
dog  is  only  slightly  susceptible,  likewise  the  cat.  Of  experimental 
animals,  mice,  guinea-pigs,  and  rabbits  are  susceptible.  Tetanus  may 
develop  in  exceptional  cases  as  a  cryptogenetic  infection,  i.  e.,  one  of 
hidden,  secret  origin,  from  the  intestinal  tract;  this  manner  of  origin, 
however,  does  not  occur  in  the  horse. 

Historical. — The  first  investigator  to  succeed  in  producing  artificial 
experimental  tetanus  was  Nicolaier  in  1885.  He  inoculated  mice, 
rabbits,  guinea-pigs,  and  dogs  with  garden  earth,  and*  by  this  means 
produced  tetanus  in  the  three  former  animals  but  not  in  the  dog.  At 
the  point  of  inoculation  he  found  slender  bacilli,  but  in  his  experiments 
he  failed  to  obtain  them  in  pure  cultures.  Rosenbach,  in  1887,  saw 
identical  bacilli  in  man  in  a  gangrenous  wound  which  had  led  to 
the  development  of  lockjaw.  Finally,  in  1889,  Kitasato  succeeded 
in  obtaining  the  tetanus  bacillus  in  pure  culture  by  raising  it  under 
strictly  anaerobic  conditions. 

Morphology  and  Staining  Properties. — The  tetanus  bacillus  when 
obtained  from  a  gelatin  culture  is  a  slender  rod  2  to  4  micra  long, 
0.3  to  0.5  micron  wide.  It  has  slightly  rounded  ends.  In  addition 
to  individual  bacilli,  chains  of  several  rods,  forming  slender  filaments, 
are  found;  the  older  the  culture  the  more  numerous  are  the  latter. 
In  older  cultures  raised  on  gelatin  at  room  temperature  many  spore- 
bearing  bacilli  are  seen.  Young  tetanus  bacilli  show  a  slight  motility, 
which  can  be  best  demonstrated  on  a  warm  stage.  The  bacilli 


270  BACILLUS  OF  TETANUS 

possess  numerous  peritrichous  flagella,  estimated  from  thirty  to  fifty 
and  even  up  to  one  hundred.  Spore  formation  in  cultures  kept 
anaerobically  in  the  incubator  occurs  after  twenty-four  to  thirty  hours; 
at  room  temperature  in  gelatin  tubes  after  eight  to  ten  days.  The 
most  abundant  sporulation  occurs  in  sugar  free  bouillon  and  on 
blood  serum.  The  tetanus  bacillus  forms  its  spore  at  one  end,  it  is 
round  and  much  larger  in  diameter  than  the  vegetative  form  of  the 
bacillus,  namely,  1  to  1.5  micra.  A  sporulating  bacillus  somewhat 
resembles  a  drum  stick  and  for  this  reason,  the  microorganism  is  also 
called  the  drum  stick  bacillus  of  tetanus.  When  the  culture  medium 
contains  sugar  or  glycerin  the  spores  are  sometimes  oval  and  not 
perfectly  spherical. 

The  tetanus  bacillus  stains  with  the  ordinary  watery  anilin  stains 
and  is  Gram  positive.  The  spores  and  the  flagella  can  only  be  stained 
by  special  methods. 

Anaerobic  Methods. — The  bacillus  is  anaerobic  and  does  not  grow  in 
the  presence  of  oxygen.  A  variety  of  methods  are  used  to  exclude 
the  atmospheric  air.  The  cultures  may  be  kept  in  an  air-tight  jar 
in  which  the  common  air  has  been  replaced  by  hydrogen  or  in  a  jar 
in  which  the  oxygen  has  been  absorbed  by  pyrogallic  acid  and 
caustic  soda  solution.  The  bacillus  may  also  be  raised  in  a  bouillon 
from  which  the  air  has  been  expelled  by  prolonged  recent  boiling  and 
the  surface  of  which  has  been  covered  by  oil  or  butter,  or  in  gelatin 
stick  cultures,  which  after  inoculation  are  covered  by  a  high  layer 
of  gelatin  containing  some  glucose. 

Cultural  Properties. — On  gelatin  plates  kept  at  20°  C.  (room 
temperature)  colonies  of  the  tetanus  bacillus  become  visible  on  the 
third  day.  The  small  young  colonies  under  a  low  magnification  show 
a  central  solid  area  surrounded  by  radiating  cords,  bands  or  filaments. 
The  young  colonies  somewhat  resemble  those  of  the  Bacillus  proteus; 
at  other  times  they  appear  more  like  those  of  the  Bacillus  subtilis.  In 
a  gelatin  stick  culture  the  growth  begins  about  one-half  inch  below 
the  surface  and  proceeds  downward,  forming  at  the  same  time  lateral 
projections  which  appear  more  or  less  cloudy.  After  the  tenth  day 
the  gelatin  becomes  more  and  more  liquefied,  and  simultaneously  a 
very  fetid  gas  is  formed.  On  agar  the  growth  is  similar  but  there  is 
no  liquefaction.  In  bouillon  kept  at  39°  C.  there  is  a  very  rapid  growth 
with  marked  clouding  and  the  appearance  of  fine  gas  bubbles. 
Sedimentation  begins  to  show  on  the  fifteenth  day.  The  gases  formed 
are  carbon  dioxide  and  ethane  and  methane  gas.  During  their 
formation  the  alkalinity  of  the  fluid  increases,  provided  no  sugar  is 
present.  The  tetanus  bacillus  does  not  grow  at  temperatures  below 
14°  C.;  at  18  to  20°  C.  the  growth  is  slow  and  easily  visible  to  the 
naked  eye  only  after  one  week;  at  20°  to  25°  C.  a  good  growth  develops 
in  three  to  four  days;  the  organism  grows  best  at  36°  to  39°  C. 

Tetanus  Bacilli  and  Aerobic  Bacteria. — The  tetanus  bacillus  when 
grown  in  artificial  pure  cultures  is,  as  has  been  stated,  strictly  anaerobic 


TETANUS  IN  THE  HORSE  AND  MAN 


271 


FIG.  138 


and  provision  must  be  made  to  exclude  the  oxygen  of  the  air.  In 
soil,  manure,  and  wounds,  however,  it  can  grow  even  without  the 
careful  exclusion  of  oxygen  when  it  is  associated  with  aerobic  micro- 
organisms, with  which  it  may  enter  into  a  symbiotic  union.  The  aerobic 
bacteria  consume  the  oxygen,  and  in  this  way  enable  the  tetanus 
bacillus  to  multiply.  On  this 
account  the  most  dangerous 
wounds  are  those  which  from 
the  beginning  have  been  con- 
taminated with  both  tetanus 
and  other  bacteria.  If  such 
wounds  are  thoroughly  cleansed 
with  antiseptics  which  destroy 
the  other  bacteria,  but  not  the 
tetanus  spores,  lockjaw  may 
yet  not  develop  in  spite  of  the 
presence  of  these  spores,  which 
do  not  readily  germinate  and 
multiply  when  alone.  It  is  also 
important  to  note  that  tetanus 
spores  which  have  been  freed 

Bacillus  tetani,  spore  formation.     (Author's 

from  the  tetanus  toxin  by  wash-  preparation.) 

ing    with    antiseptics    may    be 

taken  up  and  destroyed  by  phagocytes.  It  is  claimed  that  tetanus 
bacilli  can  be  successively  so  changed  that  they  will  grow  in  the 
presence  of  oxygen,  losing,  then,  their  toxicity  and  pathogenic  char- 
acter. This  statement,  however,  is  doubted  by  some  investigators, 
who  believe  that  these  aerobic,  non-toxic  bacilli  are  from  the  beginning 
a  pseudotetanus  bacterium. 

Resistance  of  Spores. — The  spores  are  exceedingly  resistant  to  heat 
and  antiseptics,  even  more  so  in  some  respects  than  anthrax  spores. 
They  can  survive  complete  drying  out  for  many  years;  if  raised  pri- 
marily under  the  most  favorable  conditions,  they  can,  according  to 
Smith's  experiments,  withstand  exposure  to  steam  of  100°  C.  for  one 
hour,  and  it  is  not  until  after  an  exposure  of  seventy  minutes  that  all 
tetanus  spores  are  safely  killed. 

The  Bacillus  as  a  Saprophyte. — The  tetanus  bacillus  exists  extensively 
in  the  outside  world,  particularly  in  garden  earth  and  where  there 
has  been  much  manuring;  in  warm  countries,  however,  it  is  present 
independent  of  any  manuring.  Some  investigators  believe  that  the 
tetanus  bacillus  exists  in  the  ground  only  where  the  latter  has  come  in 
contact  with  the  feces  of  horses  and  other  herbivorous  animals  which 
harbor  the  organism  in  their  intestinal  tract;  it  is,  however,  more 
generally  held  that  it  occurs  in  the  soil  independent  of  admixture 
with  fecal  matter. 

Tetanus  in  the  Horse  and  Man. — In  horses  tetanus  is  very  common 
after  nail  wounds  of  the  hoofs  and  after  castration;  in  man  after 


272  BACILLUS  OF  TETANUS 

deep  puncture  wounds  or  ragged  wounds  of  the  hands  (Fourth  of  July 
injuries).  In  both  man  and  the  horse  natural  tetanus  infection 
almost  invariably  leads  first  to  spasms  in  certain  groups  of  muscles 
and  then  progresses  symmetrically  to  the  other  muscles  of  the  body. 
The  period  of  incubation  varies  in  the  horse  from  four  to  five  days 
up  to  three  weeks.  According  to  Behring,  one  of  the  earliest  and 
most  important  symptoms  occurs  in  the  membrana  nictitans  of  the 
eye,  which,  when  the  head  of  a  horse  sick  with  tetanus  is  raised,  is 
found  to  cover  about  one-half  the  eyeball  and  to  spread  as  the  disease 
progresses.  The  head  and  neck  of  the  animal  are  elevated  until  the 
upper  border  of  the  neck  describes  a  straight  line;  in  extreme  cases  it 
is  even  concave,  and  forms  what  is  called  a  deer  neck.  Mastication  is 
difficult  in  consequence  of  the  trismus  of  the  muscles  of  mastication, 
and  finally  becomes  impossible.  The  nostrils  are  dilated.  The  tail 
in  consequence  of  the  contraction  of  its  extensor  muscles  is  stiffly  ele- 
vated. Finally  the  spinal  column  is  curved  (in  opisthotonos  position), 
and  the  muscles  of  the  neck  and  thorax  become  stiff  and  very  hard. 
All  reflexes  are  accentuated,  and  a  slight  irritation  will  bring  about 
convulsions.  Death  results  from  progressive  dyspnea.  Cattle  and 
sheep  also  suffer  from  tetanus,  but  not  nearly  as  frequently  as  horses. 

Tetanus  in  Laboratory  Animals. — The  picture  of  tetanus  is  different 
when  small  laboratory  animals,  such  as  guinea-pigs  and  rabbits, 
are  inoculated  subcutaneously  or  intramuscularly.  In  this  case  the 
contractions  begin  in  the  group  of  muscles  located  nearest  the  point  of 
injection.  If  the  inoculation,  for  example,  has  been  made  into  one  of 
the  hind  legs  it  is  the  first  to  be  affected,  and  in  succession  the  other 
hind  leg,  the  front  legs,  and,  finally,  the  muscles  of  the  back  become 
involved.  Increase  of  reflex  irritability  generally  does  not  occur, 
but  if  it  does  it  is  observed  only  shortly  before  death.  It  has  been 
noticed  that  tetanus  bacilli  when  inoculated  experimentally  do  not 
multiply  at  the  place  of  inoculation;  on  the  contrary,  soon  decrease 
in  numbers.  The  disease  is  produced  by  the  absorption  of  the 
toxins  of  the  bacillus.  Tetanus  is  one  of  the  best  examples  of  a  pure 
toxemia. 

Tetanus  Toxin. — Tetanus  toxin,  like  that  of  diphtheria,  is  soluble; 
it  easily  goes  into  solution  if  the  bacilli  are  raised  in  a  fluid  culture 
medium.  They  must,  however,  be  kept  under  strictly  anaerobic  con- 
ditions, because  only  under  these  will  they  produce  a  large  amount 
of  toxins.  If  air  is  present  the  bouillon  will  be  of  low  toxic  value. 
An  ordinary  slightly  neutral  bouillon  containing  1  per  cent,  of 
peptone  and  0.5  per  cent,  of  sodium  chloride  is  a  good  medium, 
but  it  must  contain  neither  glycerin  nor  sugar,  because  the  resulting 
acid  formation  will  interfere  with  the  toxin  production.  As  the 
latter  does  not  proceed  very  abundantly  during  the  first  few  days  of 
growth  the  cultures  must  be  kept  under  anaerobic  conditions  in  the 
incubator  for  ten  or  more  days.  The  bacteria  are  removed  from 
the  bouillon  by  filtering  it  through  a  Pasteur-Chamberland  filter 


TETANUS  TOXIN  273 

which  has  been  carefully  tested.  The  filtrate  so  obtained  is  free 
from  bacteria  and  spores,  and  contains  the  tetanus  toxin  in  solution. 
A  good  filtrate  should  contain  a  sufficient  quantity  of  a  strong  toxin, 
so  that  one  two-hundred-thousandth  part  of  a  cubic  centimeter  will 
kill  a  mouse  of  about  10  grams'  body  weight.  Some  investigators 
have  even,  by  special  means,  obtained  a  filtrate  five  times  as 
strong,  of  which  the  one-millionth  part  of  a  cubic  centimeter  would 
kill  a  mouse  of  about  10  grams'  body  weight.  By  saturating  the 
filtrate  with  ammonium  sulphate,  Brieger,  Frankel,  Buchner,  and 
others  succeeded  in  precipitating  the  toxin  in  the  form  of  a  powder, 
of  which  the  one  ten-millionth  part  of  a  gram  would  kill  a  mouse  of 
10  grams'  body  weight.  Other  animals,  however,  are  more  suscep- 
tible than  the  mouse  to  the  fatal  action  of  tetanus  poison;  the  horse, 
for  example,  is  twelve  times  as  susceptible,  and  on  the  basis  of 
the  above  figures  a  horse  weighing  2000  pounds  can  be  killed  by 
one  twelve-hundreth  gram,  or  about  one-eightieth  of  a  grain  of 
dried  tetanus  poison.  Such  powerfully  poisonous  effects  are  almost 
inconceivable.  The  tetanus  toxin  is  even  more  powerful  than  the 
venom  of  the  most  dangerous  snakes,  such  as  the  cobra.  Man  also  is 
more  susceptible  than  the  mouse,  but  probably  less  than  the  horse, 
which  is  the  most  susceptible  animal.  The  exact  susceptibility  of  man 
as  compared  with  the  mouse  and  the  horse  is  not  known,  since  it 
cannot,  of  course,  be  ascertained  by  experimental  work.  The  guinea- 
pig  is  6  times  as  susceptible  as  the  mouse;  all  other  animals  tested 
are  less  susceptible  than  the  mouse;  the  rabbit  150  times  less  sus- 
ceptible, the  goose  1000  times,  the  pigeon  4000  times,  and  the  chicken 
30,000  times.  The  latter  figure  indicates  that  for  each  gram  or 
pound  of  body  weight  of  chicken  360,000  times  as  much  tetanus 
toxin  is  needed  as  for  each  gram  or  pound  of  body  weight  of  a 
horse  in  order  to  produce  the  same  fatal  effect. 

Period  of  Incubation  of  the  Toxin. — Bacteria  and  their  toxins  never 
act  like  purely  chemical  poisons,  such  as  acids,  alkalies,  or  salts 
(strychnine  or  hydrocyanic  acid  and  its  salts,  cyanide  of  potash). 
Such  poisons  may  be  given  in  doses  large  enough  to  produce  death 
almost  instantly.  However,  if  the  most  pathogenic  bacteria  or  their 
toxins  are  inoculated  into  an  animal  even  in  very  large  doses,  death 
never  takes  place  immediately;  a  certain  period  of  time  always  elapses 
between  the  inoculation  and  the  first  symptoms.  When  tetanus  toxin  is 
inoculated  into  a  mouse  or  guinea-pig  the  period  of  incubation  becomes 
shorter  as  the  dose  is  increased;  but  it  is  impossible  to  go  beyond  a 
certain  minimum  period  of  incubation,  no  matter  how  much  the 
toxic  dose  is  increased.  If  mice  receive  3600  times  the  fatal  dose  of 
tetanus  toxin  the  period  of  incubation  is  shortened  to  a  minimum 
of  eight  hours. 

Chemistry    of   the    Toxin. — Instability. — The    chemistry    of    the 
tetanus  toxin  is  absolutely  unknown,  and  it  can  only  be  identified 
by  its  effect  upon  experimental  animals;  but  it  can,  of  course,  also 
18 


274  BACILLUS  OF  TETANUS 

be  estimated  quantitatively  by  ascertaining  the  minimum  fatal  dose 
of  a  filtrate  containing  the  tetanus  toxin.  It  must  be  understood, 
however,  that  the  toxin  is  very  unstable  in  the  watery  solution 
represented  by  the  filtrate;  it  soon  loses  in  intensity,  and  is  easily 
damaged  by  heat  and  chemicals  and  antiseptics.  The  exposure  of 
a  filtrate  containing  tetanus  toxin  to  a  temperature  of  65°  C.  for  five 
minutes  or  of  60°  C.  for  twenty  minutes  will  almost  completely 
destroy  the  toxin.  Electric  currents  passing  through  a  filtrate  will 
destroy  the  toxin  in  a  few  hours.  Mineral  acids  and  alkalies  destroy 
it  even  in  very  weak  concentration;  organic  acids  require  stronger 
concentration.  The  poison  can  easily  be  attenuated  by  the  addition 
of  certain  antiseptics.  Iodide  trichloride  (IC13),  if  present  to  the 
slight  degree  of  one  one-hundredth  per  cent,  will  in  one  hour  greatly 
attenuate  the  toxin. 

Action  of  the  Toxin  upon  the  Nervous  System. — The  effect  of  the 
tetanus  toxin  upon  the  animal  body  is  not  yet  well  understood.  It  is, 
however,  very  probable  that  the  poison  acts  upon  the  central  nervous 
system  by  uniting  with  the  cellular  elements  forming  it,  and,  further, 
that  the  toxin  travels  from  its  first  place  of  deposit  in  the  connective 
or  intramuscular  tissue  along  the  axis  cylinders  of  the  peripheral 
nerves  toward  the  central  nervous  system.  Wassermann  has  shown 
that  if  tetanus  toxin  is  mixed  with  ground-up  guinea-pig's  brain  the 
toxin  becomes  united  with  the  brain  substance,  making  it  evident 
that  an  affinity  exists  which  binds  the  tetanus  toxin  to  the  tissue  of 
the  central  nervous  system. 

Component  Bodies. — Tetanus  toxin,  as  it  is  present  in  the  filtrate, 
is  composed  of  two  bodies,  tetanospasmin,  a  substance  which  has  an 
affinity  for  and  which  affects  the  central  nervous  system  and  causes 
the  convulsions,  and  tetanolysin,  which  has  the  property  of  dissolving 
red  blood  corpuscles. 

Antitoxin  Formation. — When  tetanus  toxin  is  injected  into  an  animal 
an  antitoxin  is  produced.  The  exact  location  where  the  latter  is 
formed,  whether  in  the  central  nervous  system  or  not,  is  unknown; 
but  the  antitoxin  is  contained  in  the  blood  serum,  and  it  can  be 
obtained  by  allowing  the  blood  to  coagulate  so  that  the  serum  can 
be  separated  from  the  clot  or  coagulum.  Tetanus  antitoxin  is  manu- 
factured in  the  blood  serum  of  the  horse.  The  general  principles 
and  steps  in  forming  and  procuring  it  are  as  follows : 

1.  Inoculate  a  veal  bouillon,  containing  1  per  cent,  peptone  and 
0.5  per  cent,  sodium  chloride,  neutralized  with  carbonate  of  mag- 
nesium, and  then  slightly  acidulated  with  0.1  per  cent,  lactic  acid 
from  a  pure  culture  of  tetanus  bacilli.    Keep  under  strictly  anaerobic 
conditions  in  the  incubator  for  ten  or  more  days. 

2.  Filter  through  an  unglazed  porcelain  filter  (Pasteur-Chamber- 
land). 

3.  Ascertain  the  minimum  dose  which  will  kill  a  mouse  of  about 
10  grams  within  four  days. 


DOSAGE  275 

4.  Into  a  healthy  horse  (previously  tested  with  mallein  and  tuber- 
culin) inject  either  (a)  a  very  small  dose  of  strong  tetanus  toxin  (this 
method  is  now  little  used);  (6)  a  small  dose  of  an  attenuated  toxin 
(attenuated  with  IC13,  iodide  trichloride);  (c)  a  toxin-antitoxin  mixture 
which  is  not  completely  neutralized,  but  contains  a  small  excess  of 
toxin;  (d)  first  antitoxin  and  after  eighteen  to  twenty-four  hours,  toxin. 

5.  Repeat  injections  of  toxin  at  intervals  of  a  few  days  and  increase 
the  doses. 

6.  Draw  a  small  amount  of  blood  from  the  jugular  vein  and  test 
the  antitoxic  value  of  the  serum  on  guinea-pigs  and  mice. 

7.  After  two  to  three  months  or  longer,  when  the  antitoxic  value  of 
the  serum  is  found  to  be  high,  draw  off  (ten  to  fourteen  days  after 
last  injection)  several  thousand  cubic  centimeters  of  blood  under  the 
strictest  aseptic  precautions.     Collect  it  in  sterile  vessels  and  allow  it 
to  coagulate  in  the  refrigerator.     Separate  the  serum  from  the  clot 
and  add,  as  a  preservative,  0.5  per  cent,  carbolic  acid  or  0.4  per  cent, 
tricresol.    Distribute  into  small  dark  bottles  and  keep  in  a  cool,  dark 
place. 

Dosage. — A  good  tetanus  antitoxin  is  used  in  the  following  doses: 
Immunizing  or  preventive  dose — 10  to  20  c.c.  for  a  horse;  for  a 
smaller  animal,  5  to  10  c.c.  The  passive  immunity  conferred  upon 
an  animal  is  soon  lost.  Hence,  when  a  wound  in  a  horse,  made 
accidentally  or  by  operative  procedure,  does  not  heal  very  promptly, 
it  is  well  to  give  a  second  immunizing  injection  ten  days  after  the  first. 
A  unit  of  tetanus  antitoxin  is  approximately  ten  times  as  large  as  a 
unit  of  diphtheria  antitoxin.1 

One  unit  of  tetanus  antitoxin  is  defined  as  the  amount  of  antitoxin 
required  to  neutralize  the  effects  of  1000  times  the  minimum  dose  of 
tetanus  toxin  fatal  to  a  guinea-pig  of  350  grams.  If  the  guinea-pig  is 
protected  from  death  during  the  first  four  days  after  the  injection  of 
the  toxin-antitoxin  mixture,  which  must  have  been  prepared  fifteen 
minutes  before  the  injection,  the  dose  of  antitoxin  is  called  one  unit 
of  tetanus  antitoxin. 

The  United  States  Government  has  adopted  this  unit  of  tetanus 
antitoxin  and  provides  those  who  manufacture  antitoxin  with  a 
standardized  strong  tetanus  toxin.  The  German  standard  unit  of 
tetanus  antitoxin  is  that  amount  which  will  neutralize  the  amount  of 
a  standard  toxin  necessary  to  destroy  40,000,000  grams  of  mouse. 
The  French  standard  is  expressed  by  indicating  the  weight  of  anti- 
toxic serum  necessary  to  protect  one  gram  of  mouse  against  a  mini- 
mum fatal  dose  of  a  standard  strong  toxin.  If  one-thousandth  of  a 
gram  (0.001)  is  necessary  to  protect  a  mouse  weighing  10  grams,  one 
ten-thousandth  of  a  gram  will  protect  one  gram  of  mouse  weight;  hence, 
such  an  antitoxic  serum  would  be  called  a  1  to  10,000  antitoxic  serum. 

1  One  unit  of  diphtheria  antitoxin  is  that  dose  which  will  protect  a  guinea-pig  of  250  grams 
against  the  subcutaneous  injection  of  100  times  the  minimum  fatal  dose  of  a  strong,  fresh, 
diphtheria  toxin,  so  that  the  guinea-pig  lives  at  least  four  days,  but  will  die  after  four  days. 


276  BACILLUS  OF  TETANUS 


QUESTIONS. 

1.  What  are  the  characteristic  pathologic  lesions  of  tetanus?    What  are  the 
most  characteristic  clinical  manifestations  of  the  disease? 

2.  What  animals  are  most  susceptible  to  natural  tetanus  infection? 

3.  What  is  a  cryptogenetic  infection? 

4.  Who  discovered  the  tetanus  bacillus?  who  first  grew  it  in  pure  culture? 

5.  Describe  the  morphology  of  the  tetanus  bacillus. 

6.  What  conditions  favor  spore  formation? 

7.  What  methods  may  be  used  in  raising  tetanus  cultures  under  anaerobic 
conditions? 

8.  Describe  the  cultural  properties  of  the  Bacillus  tetani. 

9.  Discuss  the  resistance  of  the  spores  of  the  microorganism. 

10.  Where  is  the  bacillus  found  as  a  saprophyte? 

11.  Under  what  conditions  does  tetanus  frequently  make  its  appearance  in 
horses  and  human  beings? 

12.  Describe  the  early  symptoms  of  tetanus  in  a  horse. 

13.  Describe  the  formation  and  properties  of  the  tetanus  toxin. 

14.  Describe  the  process  of  separating  the  bacilli  and  their  spores  from  the  toxin 
in  order  to  obtain  a  germ-free  solution  of  the  latter. 

15.  Discuss  the  susceptibility  of  various  animals  toward  the  tetanus  toxin; 
what  animal  is  most  susceptible  to  its  poisonous  effects? 

16.  What  dose  of  tetanus  toxin  would  be  sufficient  to  kill  (a)  a  mouse;  (6) 
a  horse,  instantaneously? 

17.  What  is  meant  by  the  period  of  incubation  with  reference  to  a  toxin? 

18.  What  is  meant  by  a  single  fatal  dose  of  tetanus  toxin  for  a  mcuse  or  horse? 
What  by  a  ten  times  fatal  dose? 

19.  Give  the  exact  chemical  formula  of  tetanus  toxin  and  antitoxin. 

20.  Discuss  the  resistance  of  the  toxin. 

21.  Upon  what  structures  of  the  body  of  susceptible  beings  does  the  toxin 
act  quite  particularly?    What  is  tetanospasmin  and  tetanolysin? 

22.  Describe  the  steps  in  the  preparation  of  tetanus  antitoxin  upon  a  large 
commercial  scale. 

23.  What  is  the  immunizing  dose  of  a  strong  trustworthy  tetanus  antitoxin? 

24.  What  is  an  immunizing  unit  of  diphtheria  antitoxin?    What  of  a  tetanus 
toxin? 

25.  What  are  the  German  and  French  standards? 


CHAPTER    XXIV. 

BACILLI  OF  TYPHOID— COLON— HOG  CHOLERA  GROUP— BACILLUS 

CHOLERA  SUIS— BACILLUS  TYPHOSUS— BACILLUS  COLI  COM- 

MUNIS— WHITE  SCOURS  IN  CALVES— MALIGNANT  CATARRH 

OF     CATTLE  — BACTERIUM     PHLEGMASIA     UBERIS— 

BACILLUS  TYPHI  MURIUM— DANYSZ'S  BACILLUS 

PSITTACOSIS— BACTERIUM  PULLORUM. 

THERE  is  a  rather  large  group  of  bacilli  which  have  a  number 
.of  common  properties  and  the  phylogenetic  inter-relations  of  which 
are  evidently  comparatively  intimate.  They  do  not  resemble  each 
other  as  closely  as  the  bacilli  of  the  hemorrhagic  septicemia  group, 
but  sufficiently  to  warrant  their  classification  under  one  group.  All 
are  rather  short,  plump  rods,  they  are  generally  motile,  and  possess 
a  varying  number  of  flagella;  they  do  not  form  spores,  they  lose  the 
stain  if  treated  by  Gram's  method,  they  are  facultative  aerobics  and 
they  do  not  liquefy  gelatin.  They  differ  in  their  biologic  properties 
as  to  their  fermentative  power  toward  various  sugars  (hexoses  and 
disaccharids),  as  to  their  ability  to  form  gases,  acids,  indol,  and  a 
number  of  other  metabolic  products,  and  especially  as  to  their  patho- 
genicity  toward  man  and  the  lower  animals.  The  following  organisms, 
among  others,  belong  to  this  group:  The  bacillus  of  hog  cholera;  the 
Bacillus  coli  communis,  or  colon  bacillus;  the  bacillus  of  mouse 
typhoid;  the  bacillus  of  human  typhoid  and  the  paratyphoid  bacillus; 
the  various  dysentery  bacilli;  the  Bacillus  enteritidis;  the  Bacillus 
fsecalis  alkaligenes,  etc. 


THE  BACILLUS  OF  HOG  CHOLERA. 

Occurrence  and  Historical. — The  bacillus  of  hog  cholera,  Bacillus 
cholerse  suis,  or  Bacillus  suispestifer,  was  formerly  believed  to  be  the 
sole  cause  of  the  acute  and  highly  contagious  disease  of  swine,  known 
variously  as  hog  cholera,  swine  fever,  pneumoenteritis,  pig  typhoid, 
cholera  suum;  Schweinecholera,  or  Schweinepest  (German),  and  Pest 
du  pore  (French).  While  it  has  become  evident  that  this  disease  in 
its  pure  and  uncomplicated  form  is  due  to  a  filterable  ultramicro- 
scopic  virus  and  not  to  a  bacillus,  yet  the  latter  often  infects  swine 
during  the  course  of  hog  cholera  and  is  beyond  doubt  more  or  less 
pathogenic  for  these  animals.  The  earliest  outbreak  of  hog  cholera 
was  reported  in  Ohio  in  1833,  and  since  then  the  disease  has  spread 
over  the  entire  United  States.  It  is  believed  that  the  epidemic  was 


278  BACILLI  OF  THE  TYPHOID  GROUP 

introduced  from  Europe,  but  abroad  it  is  claimed  that  hog  cholera 
originated  in  America  and  was  from  there  transported  to  Europe. 
The  hog  cholera  bacillus  was  discovered  in  1880  by  Salmon  and 
Smith,  and  was  by  them  proclaimed  as  the  cause  of  the  disease.  The 
losses  from  this  affection  in  the  United  States  are  enormous  and  are 
estimated  at  from  ten  to  twenty-five  million  dollars  annually.  They 
are  also  very  great  in  England,  France,  Austria,  Russia,  Germany,  and 
other  smaller  European  countries. 

Pathologic  Lesions. — The  pathologic  changes  are  described  by 
Dorsett,  Bolton,  and  McBryde  as  follows:  "The  changes  seen  in  the 
internal  organs  vary  greatly  even  in  different  animals  in  one  and  the 
same  outbreak  in  a  given  herd.  In  general,  these  lesions  may  be  said 
to  be  either  those  of  a  hemorrhagic  septicemia  or  of  an  ulcerative 
enteritis,  the  latter  particularly  pronounced  in  the  cecum  and  colon. 
The  hemorrhagic  lesions  are  characteristic  of  the  rapidly  fatal  form 
of  the  disease  known  as  acute  hog  cholera,  the  ulcerative  intestinal 
lesions  being  especially  prominent  in  those  outbreaks  where  the 
animals  do  not  succumb  so  rapidly;  both  the  ulcerative  and  hemor- 
rhagic lesions  may,  however,  be  seen  in  the  same  animal.  When  the 
skin  of  the  thorax  and  abdomen  is  removed  the  subcutaneous  areolar 
tissue  may  be  found  thickly  dotted  with  ecchymoses  of  varying  size. 
In  acute  hog  cholera  the  inguinal  glands  on  both  sides  are  usually 
swollen  and  red,  the  hemorrhagic  condition  being  so  intense  at  times 
as  to  give  the  glands  a  bluish-black  color.  The  lymphatic  glands 
at  the  angles  of  the  lower  jaw  may  be  affected  in  a  similar  manner, 
as  may  also  the  bronchial,  mediastinal,  mesenteric,  mesocolic,  retro- 
peritoneal,  and  lumbar  glands.  In  the  chronic  form  of  the  disease 
the  lymph  glands  seldom  exhibit  any  change.  The  heart  frequently 
presents  at  its  outer  surface,  and  also  in  the  endocardium  at  times, 
hemorrhagic  markings.  The  lungs,  as  a  rule,  are  but  slightly  affected. 
In  the  acute  form  of  hog  cholera  they  often  show  ecchymoses  of 
varying  size  on  the  serous  surfaces;  at  times  areas  of  bronchopneu- 
monia  or  collapse  are  met  with.  In  acute  hog  cholera  the  spleen, 
as  a  rule,  is  larger  than  normal,  and  engorged  with  blood,  and  may 
present  numerous  punctiform  hemorrhages  beneath  the  capsule,  or 
larger  hemorrhagic  areas  which  are  diffuse  in  character.  In  chronic 
hog  cholera  the  spleen  may  be  smaller  than  normal,  and  in 
this  case  the  connective  tissue  is  noticeably  increased.  The  serous 
surface  of  the  stomach  may  be  flecked  with  diffuse  hemorrhages, 
and  the  mucosa  is  not  infrequently  congested  and  inflamed.  This 
inflammation  is  at  times  quite  extensive,  and  may  bring  about 
destructive  ulceration  of  the  mucous  membrane.  Small  petechise 
may  be  seen  here  and  there  over  the  mucous  membrane.  In  acute 
hog  cholera  the  chief  lesions  found  in  the  intestines  are  ecchymoses 
in  both  serous  and  mucous  coats,  together  with  erosions  of  the  mucous 
surfaces  of  both  the  large  and  the  small  bowels.  The  erosions  in 
the  cecum  and  colon  appear  to  be  the  starting  point  of  the  button-like 


THE  BACILLUS  OF  HOG  CHOLERA  279 

ulcers  which  are  frequently  encountered  in  the  chronic  form  of  hog 
cholera.  These  round  ulcers  vary  from  1  to  2  mm.  to  several  centi- 
meters in  diameter,  and  are  elevated  above  the  surrounding  healthy 
mucous  membrane.  They  are  tough  and  hard,  and  their  centres  are 
usually  dark  greenish-yellow  in  color,  and  in  the  case  of  the  larger 
ulcers,  all  four  coats  of  the  intestine  are  involved.  The  ulcers  at 
times  are  so  numerous  as  to  destroy  the  mucous  membrane,  or  at 
least  to  affect  it  over  extensive  areas  in  the  cecum  and  colon.  The 
liver  may  exhibit  extensive  fatty  degeneration  with  areas  of  coagu- 
lation necrosis  or  an  increase  of  connective  tissue.  In  the  acute 
form  of  hog  cholera,  minute  hemorrhages  may  be  visible  beneath 
the  capsule.  In  acute  hog  cholera  the  kidneys  are  practically  always 
the  seat  of  hemorrhagic  changes,  which  vary  more  or  less  in  extent. 
At  times  the  cortices  are  intensely  congested,  and  all  of  the  glomeruli 

FIG.  139  FIG.  140 


Kidneys  of  hog  in  hog  cholera.     (Dorsett,  Bolton,  and  McBryde.) 

are  visible  as  minute  deep  red  points.  In  other  instances  the  general 
congestion  is  absent,  the  major  portion  of  the  kidneys  being  rather 
paler  than  normal,  dotted  here  and  there  with  minute,  sharply  defined, 
punctate  ecchymoses.  At  times  the  medullary  portion  of  the  kidneys 
is  involved,  and  blood  clots  may  be  found  in  the  pelves.  In  chronic 
hog  cholera  these  ecchymoses  are  seldom  seen." 

In  addition  to  the  lesions  which  have  just  been  described,  the 
acute  form  may  show  nearly  all  of  the  serous  membranes  of  the  body 
dotted  with  hemorrhages.  The  blood  and  internal  organs  of  hogs 
which  have  died  of  either  acute  or  chronic  hog  cholera  usually  yield 
pure  cultures  of  the  Bacillus  cholerse  suis. 

Hog  cholera  is  conveyed  from  sick  to  healthy  animals  almost  always, 
if  not  quite  without  exception,  by  contact,  by  feeding  the  viscera  of 
diseased  animals,  and  by  the  subcutaneous  injection  of  the  blood  of 


280 


BACILLI  OF  THE  TYPHOID  GROUP 


sick  animals.  The  mortality  varies  from  30  to  100  per  cent. ;  in  the 
acute  type  the  death  rate  exceeds  80  per  cent,  of  the  affected  herd. 
Morphology  and  Staining  Properties. — While  evidently  not  the  cause 
of  the  disease,  which  can  be  transferred  from  sick  to  healthy  hogs 
by  the  filtered,  bacteria-free  blood,  the  Bacillus  suisepticus  is,  never- 
theless, in  so  close  a  relation  with  the  affection  as  it  occurs  under 
natural  conditions  that  it  must  be  studied  in  connection  with  it.  The 
bacillus  is  a  plump,  rather  short  rod,  with  rounded  ends;  it  is  from 
1.2  to  1.8  micra  long;  in  the  tissues  it  occurs  as  single  bacilli  or  in 
chains  of  two,  while  in  artificial  culture  media  it  often  forms  longer 


FIG.  141 


FIG.  142 


Bacillus  of  hog  cholera.      X  1000. 
t__  ^(Author's  preparation.) 


Bacillus  of  hog  cholera,  flagellar  stain  of 
Pittfield.  X  1000.  Cover-glass  prepared  by 
Dr.  L.  E.  Day. 


chains,  which,  however,  rarely  grow  as  long  as  those  often  formed  by 
the  typhoid  bacillus.  It  is  lively  motile,  and  surrounding  its  body  are 
from  three  to  nine  long  flagella.  It  stains  with  the  ordinary  watery 
anilin  stains,  not  so  well  with  methylene  blue,  best  with  f uchsin.  With 
the  latter  it  appears  uniformly  dyed,  with  the  former  the  centre  often 
remains  unstained.  It  is  Gram  negative.  It  does  not  form  spores. 

Cultural  and  Biologic  Properties. — The  organism  grows  at  a  wide 
range  of  temperature,  namely  between  8°  and  42°  C.,  best  at  blood 
temperature  and  in  the  presence  or  obsence  of  oxygen.  It  grows  well 
in  slightly  alkaline,  less  vigorously  in  slightly  acid  media.  It  ferments 
glucose  but  not  lactose  or  saccharose.  It  does  not  turn  milk  acid 
and  does  not  coagulate  it.  It  does  not  form  indol.  On  gelatin 
plates  round,  bluish,  transparent  colonies  are  formed,  and  in  gelatin 
stick  cultures  a  grayish-white  streak  develops.  The  medium  sometimes 
exhibits  a  milky  cloudiness  but  is  not  liquefied.  On  cigar  slants  a 
grayish-white  glistening,  not  tenacious  growth  appears  after  twenty- 
four  hours'  incubation.  On  coagulated  blood  serum  the  growth  is 
similar  to  that  on  agar.  Nutrient  bouillon  becomes  uniformly  cloudy 


THE  BACILLUS  OF  HOG  CHOLERA  281 

and  a  loose  sediment  is  soon  formed  at  the  bottom  of  the  tube.  On 
potatoes  it  either  forms  a  colorless,  almost  invisible,  moist,  slightly  shiny 
growth  or  one  which  may  show  a  pronounced  brownish  tint.  In  milk 
the  reaction  after  some  time  becomes  decidedly  alkaline  and  the  fat 
of  the  medium  undergoes  a  process  of  saponification,  while,  simul- 
taneously, the  fluid  becomes  opalescent,  thick,  and  dark  colored,  but 
not  viscid.  It  sometimes  requires  from  three  to  four  weeks  for  all 
these  characteristic  changes  to  become  manifest.  In  Dunham's 
peptone  water  the  growth  is  not  vigorous,  indol  is  generally  not  formed, 
but  occasionally  it  may  be  formed.  In  the  presence  of  glucose,  the 
bacillus,  according  to  Moore,  during  the  first  day  forms,  from  the 
sugar  in  solution,  from  one-fourth  to  one-half  of  the  total  quantity 
of  gas.  By  the  end  of  the  second  day  gas  formation  is  nearly  com- 
pleted. The  total  amount  formed  is  composed  of  carbon  dioxide  and 
hydrogen  in  the  proportion  of  one  volume  of  the  former  to  two  volumes 
of  the  latter.  In  the  presence  of  glucose  the  reaction  becomes  strongly- 
acid  and  the  development  of  the  organism  ceases.  Lactose  and  cane 
sugar  (saccharose)  are  not  fermented,  and  no  gas  is  formed  in  their 
presence. 

Resistance. — The  organism  well  resists  drying  out,  and  may  remain 
alive  in  dried-out  tissues  for  several  months.  Alternate  drying 
and  moistening,  however,  kills  it  rapidly;  also  exposure  to  direct 
sunlight.  It  may  remain  alive  for  months  in  feces  and  moist  soil; 
also  for  a  long  time  in  ordinary  water.  According  to  Preisz,  it  is 
killed  at  50°  C.  in  sixty-six  hours,  at  55°  C.  in  one  hour.  It  is  killed 
in  ten  minutes  or  less  in  1  per  cent,  carbolic  acid,  -J  per  cent,  hydro- 
chloric acid,  2^  Per  cent,  sulphuric  acid,  1  to  1000  corrosive  sublimate, 
and  1  per  cent,  milk  of  lime;  1  to  2000  formalin  solution  destroys  it 
in  three  hours. 

Animals  Susceptible. — It  is  pathogenic  in  natural  infection  to  hogs 
only,  but  mice  are  quite  susceptible  to  artificial  inoculation;  guinea- 
pigs  and  rabbits  are  less  so.  Very  large  doses  injected  intravenously 
may  kill  horses  and  cattle.  Pigs,  when  injected  subcutaneously, 
generally  develop  local  lesions  only,  but  some  fatal  cases  after  such 
injections  have  been  reported. 

The  Etiology  of  Hog  Cholera  and  the  Relation  of  the  Bacillus  Cholerae 
Suis  to  this  Disease. — Dorsett,  Bolton,  and  McBryde,  following  up  the 
earlier  work  of  de  Schweinitz  and  Dorset,  came  to  the  conclusion 
that  pure  cultures  of  the  Bacillus  cholerae  suis  injected  subcutaneously 
into  hogs  usually  produced  but  slight  disturbance,  while  a  severe 
illness  frequently  resulted  after  intravenous  injections  or  feeding. 
~  state  that  the  disease  produced  in  this  manner  may  present  the 
symptoms  and  lesions  of  acute  hog  cholera,  but  the  contagiousness  and 
the  infectiousness  of  the  blood  are  absent,  and  hogs  which  have 

covered  from  such  illness  are  not  immune  when  exposed  subse- 
[uently  to  the  natural  disease.     They,  therefore,  have  demonstrated 

tat  pure  cultures  possess  a  very  considerable  pathogenic  power  for 


282  BACILLI  OF  THE  TYPHOID  GROUP 

hogs,  and  also  that  the  disease  lacks  several  of  the  essential  features 
of  acute  hog  cholera. 

The  experiments  with  blood  serum  derived  from  hogs  sick  of  hog 
cholera  and  proved  to  be  free  from  Bacillus  cholerse  suis  show,  on 
the  contrary,  that  such  serum,  upon  subcutaneous  injection,  produces 
illness  in  hogs  with  great  regularity,  and,  furthermore,  that  the  disease 
thus  produced  possesses  all  the  characteristics  of  the  natural  disease 
including  symptoms,  lesions,  contagiousness,  infectiousness  of  the 
blood,  and  immunity  in  those  animals  which  recover.  The  striking 
contrast  of  these  results  with  those  obtained  when  cultures  of  the 
Bacillus  cholerse  suis  are  used  and  their  complete  agreement  with 
the  results  obtained  from  unfiltered  blood  of  sick  hogs  make  the 
conclusion  necessary  that  some  virus  other  than  Bacillus  cholerse  suis 
exists  in  the  blood  of  hogs  suffering  from  acute  hog  cholera,  and  that 
this  virus  is  necessary  for  the  production  of  the  disease. 

This  virus  is  only  known  by  the  effects  it  produces.  Every  attempt 
to  discover  by  microscopic  examination  or  by  the  usual  cultural 
methods  a  visible  microorganism  in  these  filtrates  has  failed  com- 
pletely. There  can  be  no  doubt  that  the  pathogenic  power  of  the 
filtered  blood  is  due  to  some  living  agent  endowed  with  the  power  of 
reproduction  and  not  to  the  presence  of  a  toxin  alone,  because  the 
disease  induced  by  the  filtered  serum  is  communicated  from  sick  to 
healthy  animals  by  association,  and,  moreover,  because  the  disease 
induced  by  filtered  serum  has  been  transferred  to  a  second  and  even 
a  third  animal  by  subcutaneous  injections,  the  serum  being  filtered 
each  time  previous  to  inoculation. 

While  experiments  by  Dorsett,  Bolton,  and  McBryde  established 
beyond  question  that  the  filterable  virus  was  present  in  all  the  out- 
breaks of  hog  cholera  studied  experimentally  by  these  authors,  it  is 
also  true  that  the  Bacillus  cholerse  suis  was  present  almost  as  uni- 
formly. Even  were  the  investigators  named  so  inclined,  it  would,  for 
this  reason,  be  impossible  to  overlook  the  part  which  this  organism 
may  have  played.  From  the  results  of  inoculations  with  the  filterable 
virus,  however,  and  those  obtained  with  cultures,  one  is  compelled 
to  conclude  that  the  prime  cause  in  these  cases  was  the  filterable 
virus,  and  that  the  Bacillus  cholerse  suis  was  at  most  an  accessory 
factor. 

The  exact  role  of  the  Bacillus  cholerse  suis  in  outbreaks  of  acute 
hog  cholera  is  difficult  to  define.  That  the  fatal  result  in  many 
instances  is  materially  influenced  by  the  presence  of  that  organism  can- 
not be  doubted;  in  addition,  the  fact  should  be  emphasized  that 
although  the  filterable  virus  appears  to  have  been  the  primary  invader 
in  the  cases  of  acute  hog  cholera  investigated,  the  possibility  of 
independent  disease  being  caused  by  Bacillus  cholerse  suis  cannot  be 
denied.  In  fact,  belief  in  such  a  possiblity  is  difficult  to  avoid  when 
the  very  considerable  pathogenic  power  for  hogs  exhibited  by  many 
cultures  of  that  organism  when  fed  or  administered  intravenously  is 
considered. 


THE  BACILLUS  OF  HOG  CHOLERA  283 

From  the  experiments  of  Dorsett  and  his  co-workers  it  became 
evident  that  the  Bacillus  cholerse  suis  was  not  the  true,  principal,  and 
sole  cause  of  hog  cholera.  Even  before  the  papers  of  de  Schweinitz 
and  Dorsett  and  his  associates  on  the  exact  etiology  of  hog  cholera 
had  been  published,  other  investigators,  such  as  Hottinger,  working  in 
Brazil,  Theiler,  in  South  Africa,  and  also  Boxmeyer  had  begun  to 
doubt  the  etiological  importance  of  the  Bacillus  suipestifer.  Hottinger 
believed  the  bacillus  to  be  an  organism  of  the  colon  croup  which 
penetrated  from  the  intestines  into  the  general  circulation,  but  which 
was  not  the  true  cause  of  the  disease.  The  experiments  of  Dorsett, 
McBryde,  Niles,  and  Bolton,  of  the  United  States  Department  of 
Agriculture  on  the  production  of  the  disease  by  bacteria-free  blood, 
were  confirmed  by  Theiler,  Hutyra,  Ostertag  and  Stadies,  McClin- 
tock,  Boxmeyer  and  Siffer,  Uhlenhut,  Xylander  and  Bohtz,  Wasser- 
mann,  Carre,  Leclainche  and  Vallee,  and  others.  There  is  at  the 
present  time  no  doubt  remaining  that  hog  cholera  is,  indeed,  due  to  a 
filterable,  invisible,  ultramicroscopic,  highly  contagious  living  virus, 
and  not  to  the  bacillus  suipestifer. 

Protective  Inoculation. — The  fact  that  animals  which  recover  from 
hog  cholera  are  immune  for  the  remainder  of  their  natural  lives  was 
noticed  early  in  the  modern  studies  of  the  disease.  Such  animals 
could  not  even  be  made  sick  by  injecting  into  them  blood  from  cholera- 
sick  hogs.  Since  the  discovery  that  the  affection  is  due  to  an  invisible 
virus,  Dorsett  has  worked  out  a  method  to  hyperimmunize  hogs,  so 
that  their  blood  contains  a  large  amount  of  antibodies  and  can  be 
used  as  an  antitoxin  or  immune  serum  to  protect  non-immune  animals 
against  a  fatal  or  serious  attack.  The  serum  is  prepared  in  a  hog 
which  has  survived  a  natural  attack  of  the  disease.  The  animal  is 
injected  with  very  virulent  blood  from  a  cholera-sick  hog,  but  it 
must  first  be  definitely  ascertained  by  the  injection  of  2  to  5  c.c. 
of  the  blood  into  two  non-immune  hogs,  that  it  actually  is  virulent. 
If  the  injected  non-immunes  do  not  become  very  sick  themselves, 
the  blood  tested  is  not  of  sufficient  virulence,  and  the  test  must  be 
repeated  until  found  satisfactory  with  virulent  blood  from  another 
source.  The  hyperimmunization  of  the  immune  hog  is  then  brought 
about  in  the  following  manner: 

1.  Subcutaneous  Injections. — (a)  Inject  the  immune  subcutaneously 
with  defibrinated  disease-producing  blood  in  the  proportion  of  10  c.c. 
for  each  pound  of  body  weight;  or  (6)  inject  the  immune  subcutan- 
eously with  1  c.c.  of  defibrinated  disease-producing  blood  for  each 
pound  of  body  weight.    After  an  interval  of  one  week  give  a  second 
injection  of  2.5  c.c.  disease-producing  blood  for  each  pound  of  body 
weight.     After  the  interval  of  another  week  give  a  third  injection  of 
5  c.c.  of  disease-producing  blood  for  each  pound  of  body  weight. 

2.  Intravenous  Injections. — (a)  Inject  the  immune  intravenously 
with  defibrinated  disease-producing  blood  in  the  proportion  of  5  c.c. 
of  blood  for  each  pound  of  body  weight;  or  (6)  inject  the  immune 


284  BACILLI  OF  THE  TYPHOID  GROUP 

intravenously  with  defibrinated  disease-producing  blood  in  the  pro- 
portion of  5  c.c.  of  blood  for  each  pound  of  body  weight,  and  after 
an  interval  of  a  week,  if  the  hog  has  recovered,  repeat  the  injection. 

3.  Intra-abdominal  Injections. — Inject  the  immune  intra-abdom- 
inally  with  defibrinated  disease-producing  blood  in  the  proportion  of 
10  c.c.  of  blood  for  each  pound  of  body  weight. 

The  danger  of  spreading  the  disease  by  immune-infected  hogs 
disappears  within  twenty-four  hours  after  an  injection,  because  after 
this  lapse  of  time,  as  has  been  shown  by  Uhlenhut,  their  drawn  blood 
cannot  transfer  the  disease;  evidently  the  virus  has  been  neutralized 
in  the  body  of  the  immune  animal.  After  an  immune  hog  has  recovered 
from  the  effects  of  the  last  injection  of  the  virulent  blood  (generally 
in  eight  to  ten  days)  its  blood  can  be  drawn  to  inject  and  immunize 
unprotected  hogs.  Blood  may  be  drawn  from  the  hyperimmunized 
animal  by  severing  the  carotid  artery  and  bleeding  it  to  death  or  by 
cutting  off  the  tail.  The  latter  method  is  preferable  for  the  first 
drawings,  as  the  bleeding  may  be  stopped  at  any  time,  thus  per- 
mitting the  immune  animal  to  live  and  to  be  used  again  to  procure 
further  supplies  of  blood.  Experiments  have  shown  that  after  hyper- 
immunization  blood  may  be  drawn  from  the  tail  three  or  four  times 
at  intervals  of  a  week,  without  perceptibly  lessening  the  antitoxic 
properties  of  the  serum  obtained.  One  week  after  the  last  collection 
of  blood  the  serum  animal  may  be  killed  by  severing  the  carotid  and 
collecting  all  the  blood  that  can  be  obtained.  The  serum  of  the 
blood  is  separated  from  the  clot  in  the  usual  manner  after  coagulation 
has  taken  place.  A  fraction  of  a  per  cent,  of  carbolic  acid  is  added 
to  the  serum.  Different  sera  obtained  are  generally  mixed  and  the 
efficacy  of  the  mixture  is  then  tested  as  follows:  Eight  young  pigs 
weighing  from  thirty  to  sixty  pounds  receive  subcutaneously  each  2  c.c. 
of  blood  from  an  acute  case  of  hog  cholera,  two  of  the  pigs  treated 
remain  unprotected  and  the  other  six  are  protected  in  groups  of  two  by 
the  injection,  respectively,  of  10  c.c.,  15  c.c.,  and  20  c.c.  of  the  immune 
serum.  A  good  immune  serum  should  protect  in  a  dose  of  15  c.c.  or 
less.  If  this  is  found  to  be  the  case,  20  c.c.  of  the  serum  is  used  as  an 
immunizing  dose  to  protect  young  pigs  weighing  from  twenty  to  one 
hundred  pounds.  The  injection  is  generally  made  on  the  inside  of 
the  hind  leg.  The  passive  immunity  so  produced  lasts  for  several 
weeks  only;  to  produce  a  more  lasting  effect,  however,  the  simul- 
taneous method  is  used  in  which  20  c.c.  of  the  immune  serum  is 
injected  on  one  side  of  the  animal,  while  the  other  receives  a  small 
amount  (about  2  c.c.)  of  virulent  blood  from  a  case  of  hog  cholera. 

Agglutination  of  Bacillus  Suipestifer  by  the  Serum  of  Hogs  Hyper- 
immunized  against  Hog  Cholera. — The  first  tests  to  ascertain  whether 
the  serum  of  hogs  sick  with  hog  cholera  would  agglutinate  the  hog 
cholera  bacillus  in  high  dilutions  were  made  by  Dinwiddie.  His 
results  were  negative.  McClintock,  Boxmeyer,  and  Siffer  found  that 
the  serum  of  normal  hogs  agglutinated  the  hog  cholera  bacillus  in  a 


BACILLUS  TYPHOSUS  285 

dilution  of  1  to  250  and  that  the  agglutinative  power  was  much  incrased 
after  inoculation  with  hog-cholera  vaccine;  it  could  be  shown  that 
the  intraperitoneal  injection  of  such  vaccines  was  usually  followed  by 
the  production  of  large  quantities  of  agglutinins.  The  amount  of 
the  vaccine,  however,  had  no  relation  to  the  amount  of  agglutinins 
formed.  Giltner  has  recently  published  a  preliminary  report  on  a 
series  of  experiments  which  show  that  the  serum  of  hogs  hyperim- 
munized  against  hog  cholera  by  the  Dorsett-Niles  method  has  a 
very  high  agglutinative  value  for  the  hog-cholera  bacillus.  He  found 
values  as  high  as  1  in  2000.  The  blood  serum  of  immune  hogs 
(which  had  not  yet  been  hyperimmunized)  agglutinated  in  dilutions 
as  high  as  1  in  1000.  These  results  are  really  not  very  astonishing 
when  the  fact  is  considered  that  hog-cholera  bacilli  are,  as  a  rule, 
found  in  the  blood  and  organs  of  animals  sick  from  hog  cholera. 
For  this  reason,  an  animal  which  survives  an  attack  of  the  disease 
may  be  expected  to  possess  a  certain  amount  of  immunity  against 
the  hog-cholera  bacillus  as  well  as  against  the  invisible  filterable 
virus.  The  presence  of  a  large  amount  of  agglutinins  against  the 
cholera  bacillus  in  hyperimmunized  animals  can  likewise  be  easily 
accounted  for  by  the  fact  that  with  the  large  amount  of  virulent, 
blood  injected  for  hyperimmunization  a  considerable  number  of 
hog-cholera  bacilli  are  also  injected.  The  agglutination  tests  with 
the  hog-cholera  bacillus  are  made  as  macroscopic  tests,  and  the 
method  is  identical  with  that  employed  in  the  case  of  the  glanders 
bacillus.  According  to  a  circular  of  the  United  States  Department  of 
Agriculture  the  Bruschettini  Hog-cholera  Vaccine  and  the  Bruschettini 
Hog-cholera  and  Swine-plague  Serum  are  without  value  whatsoever. 
The  department  reported  that  healthy  pigs  were  injected  with  the 
serum  and  were  exposed  after  twenty-four  hours  by  being  placed 
in  the  same  pens  with  hogs  affected  with  the  disease.  All  the  hogs 
treated  with  the  Bruschettini  serum  contracted  hog  cholera  within 
the  usual  period  of  time  after  exposure  and  finally  died,  exhibiting 
typical  lesions  of  hog  cholera  at  autopsy. 


BACILLUS  TYPHOSUS. 

Occurrence. — The  best  known  of  the  microorganisms  of  the  group 
under  discussion  is  the  typhoid  bacillus.  It  is  the  cause  of  typhoid 
fever,  also  called  abdominal  typhoid  or  enteric  fever  in  man.  While 
pathologic  changes  and  death  can  be  produced  in  experimental 
animals  by  the  inoculation  of  typhoid  bacilli,  none  of  the  lower 
animals  are  susceptible  to  a  natural  typhoid  infection.  In  man, 
typhoid  fever  is,  as  a  rule,  contracted  through  the  drinking  water 
or  food  which  has  directly  or  indirectly  become  contaminated  with 
the  excrements  of  patients  suffering  from  the  disease.  The  bacillus, 
after  ingestion  with  water  or  food,  multiplies  enormously  on  and  in  the 


286  BACILLI  OF  THE  TYPHOID  GROUP 

mucosa  of  the  small  intestine,  where  it  causes  local  pathologic  changes, 
such  as  swelling  and  ulceration  of  the  solitary  and  agminated  lymph 
follicles,  and  general  symptoms  due  to  the  absorption  of  its  toxins. 
At  a  later  stage  it  enters  the  general  circulation,  becomes  localized  in 
the  spleen  and  also  in  other  places,  preferably  in  the  lungs,  and  is 
then  excreted  not  merely  with  the  feces,  but  also  with  the  urine, 
sputum,  etc.  It  is  difficult  to  isolate  the  bacillus  from  the  feces,  less 
difficult  from  the  urine,  and  easiest  from  the  juice  of  the  spleen, 
provided  it  is  obtained  in  a  perfectly  aseptic  manner.  As  a  rule, 
typhoid  bacilli  disappear  from  the  urine  and  feces  within  a  few 
weeks  after  the  termination  of  the  disease;  they  generally,  how- 
ever, remain  present  for  a  long  time  in  the  gall-bladder.  In  excep- 
tional cases  persons  who  have  had  typhoid  fever  may  void  the 
bacilli  with  their  feces  for  years,  becoming  in  this  way  a  constant 
source  of  spreading  typhoid  through  contaminating  milk  or  water. 
One  attack  of  typhoid  fever,  as  a  rule,  induces  immunity  for  the 
remainder  of  the  individual's  life,  but  in  about  2  per  cent,  of  the  cases 
a  second  attack,  generally  of  a  mild  character,  has  occurred.  Oysters 
obtained  from  water  contaminated  by  sewage  may  harbor  virulent 
typhoid  bacilli,  and  when  eaten  raw,  cause  infection. 

The  Typhoid  Culture  Medium  of  Drigalski  and  Conradi. — From  a 
hygienic  standpoint  it  is  sometimes  of  great  importance  to  be  able  to 
decide  whether  drinking  water,  milk,  oysters,  etc.,  or  the  stool  of  a 
supposed  permanent  typhoid  carrier  contain  typhoid  bacilli,  and  in 
order  to  find  them  rapidly  a  special  medium  is  required.  This  is 
prepared  as  follows :  Three  pounds  of  chopped  lean  beef  are  allowed 
to  stand  with  two  liters  of  water  in  the  cold  (ice-box  in  summer)  for 
twenty-four  hours.  The  meat  infusion  is  then  boiled  for  one  hour  and 
filtered.  Add  20  grams  of  Witte's  dry  peptone,  30  grams  of  nutrose, 
and  10  grams  of  common  salt;  boil  another  hour;  filter  again.  Now 
add  60  grams  of  agar,  boil  several  hours,  neutralize  with  caustic  soda 
solution,  and  filter  clear  in  the  steam  sterilizer  or  hot-water  funnel. 
Take  300  c.c.  Kahlbaum's  litmus  solution,  add  30  grams  lactose,  and 
boil  for  fifteen  minutes.  Mix  the  fluid  agar  with  the  litmus-lactose 
solution  (the  mixture  will  generally  turn  red);  it  is  now  faintly  alkalin- 
ized  with  a  10  per  cent,  soda  solution.  Finally,  add  4  c.c.  hot  sterile 
10  per  cent,  soda  solution  and  30  c.c.  of  a  sterile  solution  (1  to  1000) 
of  crystal  violet  (Hoeschst  B).  This  medium  is  distributed  into 
sterile  test-tubes.  When  the  search  for  typhoid  bacilli  is  made,  plates 
are  poured  into  Petri  dishes  from  the  melted  medium,  and  after  the 
latter  has  set,  the  surface  is  inoculated  from  the  suspected  material. 
If  the  latter  is  a  stool,  it  must  be  diluted  with  nine  times  its  volume 
of  0.85  salt  solution.  The  plates  are  then  inverted  and  allowed  to 
stand  slightly  open  for  one-half  hour  to  permit  the  surface  to  dry 
somewhat.  They  are  then  placed  in  an  inverted  position  in  the 
incubator  and  examined  after  sixteen  to  twenty-four  hours,  when  the 
typhoid  bacilli,  if  such  have  been  present,  appear  as  small,  transparent 


BACILLUS  TYPHOSUS  287 

blue  colonies,  about  1  to  3  millimeters  in  diameter.  Colonies  of  the 
colon  bacillus  and  other  members  of  the  colon-typhoid  group  are 
larger,  coarser,  less  transparent,  and  red.  To  identify  the  bacilli 
beyond  doubt  the  small  transparent  colonies  must  be  tested  with  an 
agglutinating  typhoid  serum. 

Morphology. — The  typhoid  bacillus  is  a  rather  plump  rod  1  to  3 
micra  in  length,  0.5  to  0.8  micron  in  diameter.  It  has  rounded  ends, 
frequently  forms  longer  or  shorter  chains,  is  very  actively  motile,  and 
possesses  numerous  long,  delicate  flagella,  but  does  not  form  spores. 
It  stains  with  the  ordinary  watery  anilin  stains,  but  takes  them  rather 
slowly,  and  is  best  dyed  with  a  watery  fuchsin  solution;  it  is  Gram 
negative. 

Cultural  Properties. — The  bacillus  grows  both  in  the  presence  and 
absence  of  oxygen,  best  at  blood  temperature,  well  between  25°  and 
^7°  C.,  poorly  below  20°  C.  It  is  easily  destroyed  by  antiseptics 
and  by  heat.  It  grows  well  on  all  of  the  ordinary  laboratory  media  and 
does  not  liquefy  gelatin.  On  potatoes  it  generally  forms  an  abundant 
but  invisible  growth;  occasionally,  however,  a  heavy,  yellowish-brown, 
visible  growth.  The  typhoid  bacillus  does  not  form  gas  from  glucose, 
galactose,  or  levulose;  it  does  not  coagulate  milk. 

Formation  of  Agglutinins  and  Other  Antibodies. — When  typhoid 
bacilli  are  inoculated  into  animals,  or  when  human  beings  become 
infected  in  a  natural  way,  through  drinking  contaminated  water  or 
otherwise,  antibodies  which  will  precipitate,  agglutinate,  and  dissolve 
typhoid  bacilli  develop  in  the  blood  serum  during  the  course  of  the 
disease.  In  other  words,  the  blood  serum  then  contains  specific 
precipitins,  agglutinins,  and  bacteriolysins.  These  antibodies  appear 
comparatively  early,  and  for  this  reason  an  early  and  indubitable 
diagnosis  of  typhoid  fever  in  man  can  today,  as  a  rule,  be  established 
by  the  serum  test.  This  very  simple  test  is  usually  made  as  a  micro- 
scopic test,  and  the  only  elements  necessary  are  some  blood  from  the 
suspected  case  and  a  young,  vigorous,  typhoid-bacillus  culture.  The 
latter  is  generally  raised  on  slanted  agar  or  glycerin  agar  in  the  incu- 
bator at  blood  temperature  (37°  C).  It  is  best  to  use  a  culture  eighteen 
hours  old  for  the  test;  at  least  it  should  not  be  much  older  than 
twenty-four  hours,  because  young  cultures  contain  the  most  vigorous 
and  the  most  lively  bacilli.  Blood  is  obtained  by  puncturing  the 
cleansed  ear  or  finger  tip  of  the  patient  with  a  sharp  sterile  needle 
and  allowing  it  to  drop  (in  small  drops)  on  a  clean  slide,  so  that  the 
individual  drops  do  not  touch  but  have  a  certain  free  space  between 
them.  The  blood  is  then  allowed  to  dry  on  the  glass  slide,  which,  if  it 
is  not  to  be  used  immediately,  should  be  wrapped  in  a  clean  piece 
of  paper  and  protected  against  moisture  and  dirt.  The  test  itself 
can  be  made  at  any  time  from  this  dried  blood.  The  steps  are  as 
follows : 

1.  Remove  from  the  agar  slant  with  a  platinum  loop  some  of  the 
growth,  preferably  from  the  very  margin,  because  the  most  motile 


288 


BACILLI  OF  THE  TYPHOID  GROUP 


typhoid  bacilli  are  found  here.  Rub  up  the  loopful  of  bacteria  in  a 
clean  watch-glass  with  a  sufficient  quantity  of  distilled  water  to  make 
an  even  milky  but  not  very  concentrated  emulsion.  Place  a  small 
drop  of  this  emulsion  on  a  clean  cover-glass,  add  another  drop  of 
water  with  the  platinum  loop,  and  again  mix  thoroughly  until  an 
even,  homogeneous  emulsion  is  obtained.  Place  the  cover-glass  over 
a  concave  slide,  around  the  concavity  of  which  vaselin  has  been 
painted.  Examine  this  hanging  drop  with  oil-immersion  magnification 
to  ascertain  that  the  typhoid  bacilli  are  lively,  motile,  and  not  clumped. 
2.  Prepare  "another  cover-glass  by  placing  on  it  a  small  drop  of 
the  typhoid  bacilli  emulsion  in  the  watch-glass. 


FIG.  143 


FIG.  144 


Typhoid  bacilli  from  nutrient  gelatin. 
X  1100.     (Park.) 


Gruber-Widal  reaction.  Bacilli  gathered  into 
one  large  and  two  small  clumps,  the  few  isolated 
bacteria  being  motionless  or  almost  so. 


3.  Mix  a  small  drop  of  dried  blood  on  the  cover-glass  with  about 
10  to  20  drops  of  distilled  water,  which  are  added  with  the  platinum 
loop.    Then  rub  up  well  with  the  dried  blood,  which,  of  course,  is  now 
dissolved  out  by  the  water.    This  mixing  is  done  directly  on  the  slide 
on  which  the  blood  was  collected  and  allowed  to  dry. 

4.  Add  to  the  emulsion  on  the  second  cover-glass,  with  platinum 
loop,  a  small  drop  of  the  dilute  blood,  mix  well,  and  place  cover-glass 
on  a  concave  slide. 

There  are  now  two  hanging-drop  preparations:  the  first  one  is 
simply  an  emulsion  of  young  typhoid  bacilli  in  distilled  water,  which 
serves  as  a  control  for  the  second,  which  contains  bacilli  and  distilled 
water  together  with  diluted  blood  from  the  suscepted  cases  of  typhoid 
fever.  If  this  case  is  indeed  one  of  typhoid  fever  it  will  be  noted 
after  fifteen  to  thirty  minutes  (sometimes  even  earlier)  that  the  typhoid 
bacilli  mixed  with  the  dilute  blood  lose  their  motility,  become  glued 
to  each  other  in  little  masses,  and  finally  become  indistinct,  while 


BACILLUS  COLI  COMMUNIS  289 

some  of  them  are  actually  dissolved.  This  process  is  spoken  of 
as  the  immobilization,  agglutination,  and  solution  of  the  bacilli. 
This  serum  test  is  known  as  the  Gruber-Widal  test.  The  identical 
principle  is  employed  in  a  microscopic  or  a  macroscopic  test  in 
glanders,  although  in  this  case  much  higher  dilutions  must  be  used 
(see  chapter  on  Glanders).  The  test  and  control  test  are  frequently 
made  on  a  single  slide  with  two  concavities.  The  hanging  drop  with 
water  only  is  placed  over  one  and  the  hanging  drop  containing  the 
dilute  blood  serum  over  the  other.  In  making  the  test  in  the  manner 
described  above,  care  must  always  be  taken  to  have  young  motile 
typhoid  bacilli  and  to  dilute  the  blood  sufficiently.  If  too  strong  a 
concentration  of  the  human  serum  is  used,  a  certain  amount  of 
immobilization  and  agglutination  will  occur,  and  this  may  simulate  a 
positive  reaction.  The  student  must  also  remember  that  little  masses 
of  blood  corpuscles  or  fibrin  are  found  in  dried  and  redissolved  blood ; 
these  must  not  be  mistaken  for  agglutinated  bacilli.  In  a  dilution  of 
1  in  20  of  the  dried  blood  the  reaction  should  begin  to  manifest 
itself  in  about  twenty  minutes;  in  a  dilution  of  1  in  10  in  less  than 
fifteen  minutes,  often  in  five  minutes.  When  the  reaction  with  the 
1  in  10  dilution  is  doubtful  lesser  dilutions  should  always  be  made. 
A  positive  test  is  not  found  during  the  very  first  days  of  the  disease, 
but  about  20  per  cent,  of  cases  of  typhoid  give  a  positive  reaction 
during  the  first  week  and  90  per  cent,  in  the  fourth  week.  Some  cases 
of  typhoid  never  give  a  positive  reaction  either  during  or  after  the 
disease.  The  author  has  seen  one  case  in  which  a  positive  reaction 
was  only  obtained  on  a  single  day  during  the  course  of  the  disease 
(third  or  fourth  week),  never  before,  never  afterward.  Sometimes  a 
positive  reaction  persists  for  years  after  the  disease  has  run  its  course 
and  some  permanent  typhoid  carriers  may  give  a  positive  reaction  as 
long  as  they  harbor  bacilli. 

The  Widal-Gruber  test  for  typhoid  fever  has  been  used  in  hundreds 
of  thousands  of  cases  throughout  the  world.  If  made  with  the  neces- 
sary precautions  it  is  a  most  excellent  and  trustworthy  test,  but  it 
is  not  absolutely  infallible  on  each  and  every  application. 

The  agglutination  test  in  typhoid  fever  has  been  so  fully  explained 
in  all  its  details  because  it  is  the  best  known  and  most  extensively 
used  microscopic  agglutination  test.  Its  principle  and  technique  is 
applicable  to  many  microbic  infections  in  animals,  perhaps  less  in 
practice  but  extensively  in  scientific  laboratory  work  in  the  investi- 
gation of  problems  of  immunization  in  animal  diseases. 


BACILLUS  COLI  COMMUNIS. 

Occurrence. — The  Bacillus  coli  communis,  or  Bacillus  coli  or  colon 
bacillus,  was  first  found  in  Naples  by  Emmerich  in  the  organs  and 
blood  of  patients  who  had  died  of  Asiatic  cholera.    Emmerich  thought 
19 


290  BACILLI  OF  THE  TYPHOID  GROUP 

that  he  had  discovered  the  cause  of  this  disease,  and  named  the  micro- 
organism Bacillus  Neapolitanus.  In  the  following  year  Escherich 
demonstrated  the  bacillus  in  the  stools  of  normal  milk-fed  infants, 
and  it  has  since  been  found  as  an  entirely  normal  inhabitant  in 
the  intestines  of  man  and  most  of  the  domestic  and  wild  animals. 
It  is,  however,  claimed  that  the  bacillus  does  not  occur  in  the  intes- 
tines of  the  horse.  The  organism  is  found  widespread  in  nature  as 
a  saprophyte.  Some  authors,  like  Fliigge,  strongly  maintain  that  the 
organism  is  ubiquitous  and  occurs  extensively  in  air,  soil,  water,  and 
independent  of  contamination  from  human  feces  or  animal  manure. 
Escherich  and  Pfaundler,  conceding  its  widespread  prevalence  in 
the  outside  world,  incline  to  the  belief  that  it  is  generally,  directly 
or  indirectly,  derived  from  fecal  matter.  They  admit,  however,  that 
the  bacillus  has  also  been  found  in  moderate  numbers  in  water,  which, 
according  to  chemical  analysis,  was  absolutely  pure  and  unobjection- 
able and  showed  no  chemical  evidence  of  contamination  with  sewage. 
Weisenfeld,  in  56  specimens  of  water,  both  good  and  bad,  always  found 
the  Bacillus  coli,  and  claims,  for  this  reason,  that  the  presence  of  this 
organism  cannot  be  used  as  an  index  of  contamination  with  sewage. 
The  same  standpoint  was  previously  taken  by  Kruse,  Beckmann, 
Pajal,  Miquel,  Schumann,  and,  as  stated,  by  Fliigge.  The  question 
has  been  here  discussed  because  occasionally  it  becomes  a  matter  of 
dispute  whether  milk  and  other  foods  containing  the  colon  bacillus 
must  be  considered  as  contaminated  with  fecal  matter  or  not.  There 
is  no  doubt  that  enormous  numbers  of  the  colon  bacillus  occur  in  the 
intestines  of  cattle,  as  it  is  found  in  great  numbers  in  the  intestines  of 
man.  In  both  man  and  animals  the  bacillus  is  ordinarily  not  patho- 
genic, but  in  fact  beneficial.  It  cannot  split  up  and  produce  putre- 
factive changes  in  native  albumins,  but  it  splits  up  carbohydrates 
(starches  and  sugars).  It  is  quite  evident  that  it  plays  a  physiologic 
and  beneficial  part  in  the  intestines  and  prevents  excessive  putrefactive 
processes.  While  actual  counts  of  the  colon  bacillus  in  the  feces  of 
animals  have  not  been  made,  it  has  been  ascertained  by  Eberle  and 
Lange  that  1500  to  3500  millions  per  gram  of  feces  are  present  in  the 
stool  of  milk-fed  healthy  infants,  and  according  to  Sucksdorf 's  estima- 
tion the  figure  in  adults  is  still  381,000,000  per  gram.  Ordinarily  the 
Bacillus  coli,  as  stated,  is  not  pathogenic,  but  it  may,  in  consequence  of 
inflammatory  processes,  leave  the  lumen  of  the  intestines  and  wander 
through  the  damaged  wall  of  the  gut  into  the  peritoneal  cavity,  the 
bladder,  the  ovary,  the  kidneys,  etc.,  producing  either  alone  or  in 
combination  with  other  bacteria  serious  pathologic  conditions. 

Morphology  and  Staining  Properties. — The  Bacillus  coli  is  generally 
a  short,  rather  plump  rod,  2  to  4  micra  long  (sometimes  as  long  as 
6  micra),  and  from  0.4  to  0.7  micron  wide,  with  rounded  ends.  In 
culture  media  and  also  in  tissues  which  it  has  invaded  in  a  patho- 
genic manner  it  sometimes  becomes  very  short,  so  that  it  resembles  a 
coccus.  It  occurs  singly  or  in  pairs,  and  in  culture  media  also  in  longer 


BACILLUS  COLI  COMMUNIS 


291 


chains.  It  does  not  form  spores,  but  sometimes  has  a  capsule;  it  is 
rather  sluggishly  motile,  and  possesses  flagella,  generally  four  to  eight, 
sometimes  as  few  as  two,  but,  according  to  Loeffler,  often  as  many  as 
ten  to  twenty.  The  flagella  are  generally  shorter  and  thinner  than 
those  of  the  hog  cholera  bacillus.  The  bacillus  stains  with  the  ordinary 
watery  anilin  stains,  best  with  fuchsin;  it  sometimes  shows  deeper 
stained  polar  granules  and  unstained  portions  in  the  centre.  It  is 
Gram  negative,  like  all  the  other  bacilli  of  the  hog-cholera-typhoid- 
colon  group. 


FIG.   145 


FIG.  146 


Bacillus  coli  communis.      X  1000. 
(Author's  preparation.) 


Shiga's  dysentery  bacillus.      X  1000. 
(Author's  preparation.) 


Cultural  Properties. — It  grows  best  aerobically,  but  also  anaerobically 
at  temperatures  from  10°  to  37°  C.,  and  particularly  well  at  blood 
temperature.  On  gelatin  plates  the  colonies  appear  after  eighteen  to 
twenty-four  hours.  They  are  grayish  white,  opaque,  and  moist;  in 
the  depth  of  the  medium  they  are  finely  granular  and  yellowish ;  later 
they  become  larger,  more  coarsely  granular,  and  darker.  They  are 
round,  oval,  or  whet-stone  like.  JThe  gelatin  is  not  liquefied.  In 
stick  cultures  the  development  is  *  nail-like,  and  the  surface  growth 
becomes  relatively  abundant,  covering  the  whole  surface.  On  agar 
the  organism  forms  a  grayish-white,  rather  moist  growth.  It  clouds 
nutrient  bouillon  rapidly,  and  sometimes  forms  a  pellicle.  On  potatoes 
an  abundant  development  rapidly  occurs;  it  is  yellowish  and  moist, 
and  markedly  elevated.  The  growth  on  coagulated  blood  serum  is 
much  like  that  on  agar.  Milk  is  coagulated  in  consequence  of  the 
formation  of  lactic,  acetic,  formic,  and  succinic  acids.  The  colon 
bacillus  ferments  glucose,  lactose,  maltose,  saccharose  as  well  as 
other  carbohydrates,  and  in  their  presence  forms  carbon  dioxide  and 
hydrogen,  generally  in  the  proportion  of  one  to  two.  Some  varieties 
of  the  colon  bacillus  vary  considerably  as  to  the  amount  and  proportion 


292  BACILLI  OF  THE  TYPHOID  GROUP 

of  gas  formation.  In  Dunham's  peptone  solution  the  bacillus  forms 
indol.  The  term  Bacillus  coli  does  not  cover,  as  it  is  becoming 
more  and  more  evident,  a  single  species,  but  a  number  of  closely 
allied  varieties. 


WHITE  SCOURS,  OR  DIARRHEA,  IN  CALVES. 

Occurrence  and  Historical. — White  scours,  or  diarrhea,  in  calves, 
dysenteria  neonatorum,  diarrhoea  neonatorum;  "Ruhr  der  Sauglinge," 
"Durchfall  des  Sauglinge,"  "Kalberruhr"  (German),  is  an  acute, 
contagious  diarrhea  of  calves,  attacking  them  during  the  first  days 
of  their  lives.  The  disease  has  occasionally  also  been  observed  among 
foals,  lambs,  and  pigs.  The  disease  in  calves  is  generally  caused  by 
bacteria*  representing  different  varieties  of  the  colon  bacillus.  This 
was  established  through  the  long-continued  investigations  and  experi- 
ments of  Jensen,  who  was  the  first  to  make  a  bacteriologic  study 
of  the  disease  (1891).  His  early  findings  of  pathogenic  colon  bacilli 
in  the  blood  and  organs  of  calves  dead  from  the  disease  was  first 
confirmed  by  Pina,  Monti,  and  Veratti. 

The  disease  generally  begins  one  to  two  days  after  birth,  sometimes 
within  a  few  hours  after  delivery.  After  two  to  four  further  days  the 
clinical  picture  of  the  disease  is  well  developed. 

Pathologic  Lesions. — Two  forms  of  the  disease,  varying  in  rapidity 
of  the  course  and  the  pathologic  lesions,  can  be  distinguished.  In  the 
rapidly  fatal  form  the  mucosa  of  the  stomach  and  intestines  is  red 
and  hyperemic,  hemorrhages  are  seen  here  and  there,  and  the  contents 
of  the  intestines  are  hemorrhagic.  The  peritoneal  covering  of  the 
intestines  is  likewise  hyperemic.  The  mesenteric  glands  are  swollen, 
hyperemic,  and  edematous.  The  spleen  is  generally  enlarged,  some- 
times considerably.  In  the  blood,  colon  bacilli  are  found  in  consider- 
able numbers.  In  the  second  type,  which  generally  appears  somewhat 
later  after  birth,  and  which  takes  a  somewhat  slower  course,  the 
hyperemia  of  the  gastro-intestinal  tract  and  of  the  internal  organs  in 
general  is  not  so  great,  but  of  a  rather  moderate  degree.  The  intestines 
are  flabby,  pale,  extended  by  gas;  the  intestinal  mucosa  is  only  very 
moderately  hyperemic.  The  mesenteric  glands  are  swollen,  but 
pale;  the  spleen  is  not  swollen.  Colon  bacilli  are  not  found  in  the 
blood,  but  in  the  intestines  only. 

Diarrhea  in  calves  has  also  been  sometimes  ascribed  to  bacilli  of 
the  colon  group,  which  are  more  nearly  related  to  the  hog  cholera 
than  to  the  colon  bacillus.  Jensen  also  observed  some  cases  of 
diarrhea  in  calves  which  he  considered  due  to  an  infection  with  the 
Bacillus  proteus  vulgaris.  All  these  cases,  except  those  apparently 
due  to  pathogenic  varieties  of  the  colon  bacillus,  however,  are  rare; 
the  latter  have  also  been  designated  as  "coli  bacillosis  of  calves." 
Moore,  likewise,  has  found  a  variety  of  the  colon  bacillus  as  the 


BACILLUS  TYPHI  MURIUM  293 

cause  of  diarrhea  in  calves,  while  Nocard,  who  studied  an  epidemic 
of  white  scours  among  calves  in  Ireland,  attributed  it  to  a  bacillus  of 
the  hemorrhagic  septicemia  group. 

MALIGNANT  CATARRH  OF  CATTLE. 

This  disease,  also  known  as  rhinitis  gangrsenosa,  "Bosartige  Kopf- 
krankheit  (German),  mal  de  tete  de  contagion  (French),  is  an  acute, 
non-contagious  disease  of  cattle  and  buffaloes,  involving  particularly 
the  mucous  membranes  of  the  head  in  which  pseudomembranes  and 
ulcerations  accompanied  by  profound  nervous  disturbances  are 
produced.  The  disease  has  been  described  in  most  countries  of 
Europe  and  in  South  Africa  in  cattle,  and  in  India  and  Java  in  water 
buffaloes  or  carabaos.  Leclainche,  in  1898,  reported  a  bacterium  with 
the  characters  of  the  colon  bacillus  as  the  cause  of  the  disease;  in 
experimental  work  the  organism  proved  to  be  pathogenic  for  cattle, 
rabbits,  and  guinea-pigs.  The  French  investigator  found  these 
virulent  bacilli  in  the  intestines  and  mesenteric  glands,  and  sometimes 
also  on  the  nasal  mucous  membrane,  the  papillae  of  the  tongue,  and 
the  sublingual  glands.  The  bacillus  isolated  by  Leclainche  evidently 
forms  a  very  toxic  substance  in  bouillon  cultures,  and  if  2  c.c.  of  such 
a  growth  is  injected  into  young  cattle,  profound  but  transitory  nervous 
symptoms,  such  as  restlessness,  trembling,  elevation  of  temperature, 
colicky  pains,  running  from  the  nose,  and  watering  of  the  eyes  are 
produced.  Leclainche's  investigations  have  not  yet  been  confirmed 
by  others. 

BACTERIUM  PHLE6MASIA  UBERIS. 

This  name  was  given  by  Kitt  to  a  bacillus  which  he  isolated  in 
1886  from  cases  of  inflammation  of  the  udder  in  cows  (parenchymatous 
mastitis).  It  is  now  known  that  the  bacterium  of  Kitt  is  identical 
with  the  colon  bacillus. 

BACILLUS  TYPHI  MURIUM. 

This  organism,  also  known  as  the  bacillus  of  mouse  typhoid,  first 
isolated  from  the  blood  of  mice  by  Loeffler,  is  a  member  of  the  colon 
group.  It  grows  in  the  presence  or  absence  of  oxygen,  stains  well 
with  the  watery  anilin  stains,  but  not  by  Gram's  method;  it  does  not 
liquefy  gelatin,  and  forms  acid  in  milk  without  coagulating  the  medium. 
The  organism  is  not  pathogenic  to  the  ordinary  domestic  animals, 
either  mammals  or  birds.  It  kills  mice,  however,  in  from  eight  to 
ten  days  when  introduced  into  their  intestinal  canal;  when  injected 
subcutaneously  it  kills  them  in  one  or  two  days.  The  bacillus  has 
been  used  a  number  of  times  to  rid  large  areas  of  mice;  the  most 
successful  attempt  of  this  kind  was  made  by  Loeffler  in  Greece,  where 
he  succeeded  in  ridding  the  country  of  a  pest  of  field  mice. 


294  BACILLI  OF  THE  TYPHOID  GROUP 


BACILLUS  OF  DANYSZ. 

A  bacillus  either  very  similar  to  or  identical  with  the  Bacillus 
typhi  murium  was  discovered  by  Danysz,  and  is  known  by  the  name 
of  the  discoverer.  It  has  been  much  exploited  commercially  as  a 
destroyer  of  rats.  According  to  Rosenau's  experiments  the  so-called 
Danysz-virus  and  similar  preparations  are  worthless  in  the  destruc- 
tion of  rats. 


PSITTACOSIS  OR  SEPTICEMIA  OP  PARROTS. 

This  disease  of  African  and  American  species  of  parrots,  also  called 
mycosis  of  parrots,  is  of  interest  because  it  is  claimed  that  it  is  com- 
municable to  man,  producing  in  persons  exposed  to  the  contagion  a 
frequently  fatal  type  of  pneumonia.  The  disease  in  parrots  is  char- 
acterized by  prostration  and  diarrhea,  and  in  its  course  small  grayish- 
wnite  nodules  are  formed  in  the  liver  and  other  internal  organs.  The 
bacteriology  of  the  disease  has  been  studied  by  Nocard,  who  describes 
a  bacterium  belonging  to  the  colon  group  as  its  cause.  It  grows 
both  aerobically  and  anaerobically  on  the  ordinary  culture  media. 
In  bouillon  it  grows  rapidly  in  the  incubator,  clouds  the  medium 
uniformly,  and  forms  a  thin  surface  pellicle  which  sinks  to  the  bottom 
of  the  tube  on  slight  agitation.  On  gelatin  it  forms  light  bluish, 
shining,  and  also  darker  opaque  porcelain  white  colonies.  It  grows 
also  on  agar,  on  potatoes,  in  milk.  Gelatin  is  not  liquefied,  lactose 
not  fermented,  and  milk  not  coagulated  by  it.  The  bacillus  of 
psittacosis,  therefore,  in  cultures  acts  more  like  the  typhoid  or  hog- 
cholera  bacillus  than  the  Bacillus  coli.  According  to  Dupuy's 
statistics,  as  quoted  by  Nocard,  seventy  persons  living  in  Paris  during 
the  years  1892  to  1897  contracted  psittacosis  from  parrots,  and 
twenty-four  of  these  died.  It  is  believed  that  the  disease  is  generally 
contracted  by  persons  kissing  the  sick  parrots  or  by  otherwise  coming 
in  too  close  contact  with  them.  The  first  cases  of  psittacosis  pneumonia 
in  man  were  probably  observed  by  Ritter  in  Switzerland  in  1879. 
Palamidessi  observed  five  cases  in  one  family  in  Florence.  Leich- 
tenstern,  who  saw  some  cases  of  this  peculiar  pneumonia  in  man  in 
Germany,  doubts  that  they  have  their  origin  in  parrots  suffering 
from  psittacosis. 

BACTERIUM  PULLORUM. 

Occurrence. — An  epidemic  disease  with  a  very  high  mortality  occurs 
among  very  young  chicks  recently  hatched.  The  young  fowls  first 
show  a  loss  of  appetite  and  sluggishness,  the  feathers  become  ruffled, 
and  diarrhea  appears;  the  droppings  are  of  a  whitish  color.  The 
disease  is  known  under  the  name  of  white  diarrhea  of  chicks.  As 


BACTERIUM  PULLORUM  295 

the  cause  of  this  affection,  Rettger  and  Harvey  have  described  a 
specific  organism,  which,  according  to  its  morphologic  and  cultural 
properties,  belongs  to  the  colon-hog-cholera  group  of  bacteria. 

Pathologic  Lesions. — The  chicks  on  postmortem  examination  are 
found  to  be  much  emaciated;  the  crop  is  empty,  the  intestines  are  pale, 
but  without  indications  of  ulceration  or  congestion,  and  the  liver  is 
pale,  with  the  exception  of  a  few  patches  and  streaks  which  are  of  a 
dark  color,  while  the  spleen,  lungs,  and  kidneys  appear  normal.  In 
stained  sections  of  the  tissues  small  slender  bacilli  are  occasionally 
found.  These  organisms  do  not  occur  in  groups,  but  singly,  scattered 
here  and  there  through  the  section.  While  in  healthy  chicks  or  in 
those  which  have  died  from  other  causes  the  yolk  is  generally  com- 
pletely absorbed  at  this  age,  in  chicks  dead  from  white  diarrhea 
the  yolk  sacs  are  not  yet  absorbed,  but  are  present,  varying  in  size  from 
a  small  pea  to  an  Italian  chestnut.  According  to  Rettger  and  Harvey 
the  best  method  of  obtaining  the  organism  in  first  culture  is  to  open 
the  body  with  a  sterile  knife,  then  remove  as  much  blood  as  possible 
with  a  sterile  platinum  loop;  also  pieces  of  spleen  or  liver,  or  some 
•of  the  contents  of  the  unabsorbed  yolk  sac,  and  make  streaks  on  agar 
slants.  The  latter  are  to  be  incubated  at  35°  to  37°  C.  In  the  course 
of  twenty-four  hours,  inspection,  preferably  with  a  hand  magnifying 
lens,  will  reveal  the  presence  of  minute  colonies  which  resemble  small 
droplets  of  fat.  The  colonies  are  discrete,  and  remain  so  even  after 
several  days  of  incubation.  The  colonies  never  grow  to  be  large, 
although  there  is  considerable  increase  in  size  after  the  first  twenty- 
four  hours.  The  growth  on  agar  streaked  with  the  infected  blood 
during  the  first  twenty-four  to  thirty-six  hours  has  all  the  appearances 
of  the  ordinary  streptococcus. 

Morphology. — The  Bacterium  pullorum,  isolated  by  Rettger  and 
Harvey  from  white  diarrhea,  or  septicemia  of  chicks,  is  a  long,  slender 
bacillus,  3  to  5  micra  long  by  1  to  1.5  micra  wide,  with  slightly  rounded 
ends.  It  usually  occurs  singly,  chains  of  more  than  two  bacilli  being 
rarely  found.  It  is  non-motile,  and  resembles  the  bacillus  of  typhoid 
fever.  It  is  stained  readily  by  the  ordinary  watery  anilin  stains,  and 
is  Gram  negative.  It  does  not  form  spores. 

Cultural  Properties. — The  colonies  on  agar  slants  have  been  already 
described.  On  gelatin  plates  it  forms  surface  colonies  which  some- 
what resemble  the  grape-leaf  colonies  of  the  typhoid  bacillus.  In 
gelatin  stick  cultures  a  delicate  growth  appears  in  forty-eight  hours 
along  the  whole  line  of  inoculation;  the  growth  is  distinctly  granular 
in  appearance,  and  spreads  very  little  on  the  surface.  Gelatin  is 
not  liquefied.  The  development  on  potato  is  slow;  in  litmus  milk 
there  is  no  change  for  forty-eight  hours,  then  the  medium  becomes 
slightly  acidified,  but  there  is  no  coagulation.  The  organism  can  split 
dextrose  and  mannite  with  acid  and  gas  production,  but  it  does  not 
ferment  either  maltose,  lactose,  saccharose,  inulin,  or  dextrin.  It 
does  not  form  indol  or  nitrite  in  Dunham's  solution. 


296  BACILLI  OF  THE  TYPHOID  GROUP 

Rettger  and  Harvey  inoculated  a  number  of  chicks  with  three  stems 
of  their  bacillus  obtained  from  three  different  epidemics.  Most 
inoculations  were  successful  and  led  to  a  typical  fatal  attack  of 
white  diarrhea.  From  the  dead  chicks  the  organism  could  again 
be  obtained  in  pure  cultures.  A  few  feeding  experiments  also  gave 
positive  results. 

Morse,  who  studied  white  diarrhea  in  chicks  before  the  publication 
of  Rettger  and  Harvey's  work,  came  to  the  conclusion  that  the  affection 
was  due  to  a  protozoan  organism,  i.  e.,  Coccidium  tenellum. 

QUESTIONS. 

1.  What  bacteria  belong  to  the  typhoid-colon-hog  cholera  group  of  bacilli? 

2.  Name  some  of  their  common  properties. 

3.  In  what  respect  do  they  differ  from  each  other? 

4.  What  other  names  have  been  given  to  the  disease  hog  cholera? 

5.  What  is  the  hog-cholera  bacillus  or  Bacillus  suipestifer? 
,     6.  Discuss  the  geographical  occurrence  of  hog  cholera. 

7.  Describe  in  general  terms  the  pathologic  lesions  of  the  disease. 

8.  Describe  the  lymphatic  glands  in  the  disease;  also  the  changes  found  in 
the  gastro-intestinal  tract. 

,     9.  Describe  the  appearance  of  the  kidneys. 

10.  Describe  the  morphology  and   the  staining  properties  of  the   Bacillus 
cholerae  suis. 

11.  At  what  temperature  does  this  organism  grow? 

12.  What  sugars  are  fermented  by  this  bacillus? 

13.  Describe  its  growth  on  gelatin  plates  and  in  gelatin  stick  culture. 

14.  How  does  it  act  when  growing  in  milk?    How  in  bouillon? 

15.  Does  it  form  indol? 

16.  Give  the  differential  features  of  Bacillus  suipestifer  and  Bacillus  (bipolaris) 
suisepticus. 

17.  Discuss  the  resistance  of  Bacillus  cholerae  suis. 

18.  Give  a  definition  of  the  term  filterable,  invisible,  ultramicroscopic  virus. 

19.  Describe  the  experiences  and  experiments  which  have  led  to  the  recog- 
nition of  the  fact  that  hog  cholera  is  not  due  to  the  Bacillus  cholerae  suis. 

•  20.  Describe  the  method  of  hyperimmunizing  hogs  against  hog  cholera  to 
obtain  an  immune  or  antiserum  of  high  value. 

21.  What  is  an  immune  hog? 

22.  How  is  hog-cholera  blood  tested  as  to  its  virulency? 

23.  How  soon  is  the  virulent  blood  neutralized  in  the  body  of  an  immune  hog? 

24.  Describe  methods  of  obtaining  the  immune  serum  from  the  hyperimmunized 
hog. 

25.  Describe  the  method  of  testing  the  immunizing   or  protecting   power  of 
the  immune  serum. 

26.  Describe  the  method  of  procuring  passive  immunity  in  non-immunes. 

27.  Describe  the  simultaneous  method  of  producing  a  more  lasting  active 
immunity. 

28.  In  what  dilution  does  the  blood  serum  from  a  healthy  hog  agglutinate  the 
hog-cholera  bacillus? 

'  29.  What  effect  has  the  intraperitoneal  injection  of  vaccines  prepared  from 
the  Bacillus  cholerae  suis  upon  the  agglutinating  power  of  hog's  blood  toward 
this  organism? 

30.  How  does  the  blood  serum  of  hogs  hyperimmunized  against  hog  cholera 
behave  toward  the  Bacillus  cholerae  suis? 

31.  What  human  and  what  animal  diseases  are  caused  by  the  Bacillus  typhosus? 

32.  How  is  typhoid  fever  spread? 

33.  How  can  typhoid  bacilli  be  detected  in  milk  or  water? 

34.  Describe  the  preparation  of  the  Drigalski-Conradi  medium. 
"35.  How  is  it  used?    How  do  typhoid  cultures  look  on  it? 

36.  Describe  the  morphology  of  the  typhoid  bacillus. 


QUESTIONS  297 

37.  Describe  its  cultural  properties. 

38.  How  does  it  generally  grow  on  potatoes? 

39.  How  does  it  act  toward  milk;  toward  different  sugars  in  solution  in  the 
culture  media? 

40.  What  kind  of  immune  bodies  (antibodies)  are  formed  in  typhoid  infection 
in  man  and  animals? 

41.  What  is  the  Gruber-Widal  serum  test  for  typhoid  fever? 

42.  What  apparatus,  cultures,  reagents  are  necessary  to  make  the  test? 

43.  What  kind  of  a  culture  is  to  be  used;  how  is  the  blood  to  be  obtained  and 
treated? 

44.  Describe  in  detail  the  steps  to  make  the  test.    What  is  the  outcome  in  a 
positive,  and  what  in  a  negative  test? 

45.  Discuss  the  advantages  and  the  shortcomings  of  the  test. 

46.  What  is  meant  by  the  term  permanent  typhoid  bacilli  carrier?  (The  German 
word  for  this  is  "Dauerausscheider.") 

47.  Why  should  the  veterinary  student  become  familiar  with  the  technique 
of  the  Gruber-Widal  test  in  typhoid? 

48.  Give  the  other  names  in  use  for  the  colon  bacillus. 

49.  Where  is  this  bacillus  found  in  connection  with  animal  life?    Where  is  it 
found  in  the  outside  world? 

50.  Is  its  presence  always  indicative  of  contamination  with  feces,  manure,  or 
sewage?    What  are  the  views  held  by  different  authors  on  this  point? 

51.  Under  what  conditions  may  the  colon  bacillus  become  pathogenic? 

52.  Describe  the  morphology  of  the  bacillus. 

53.  Describe  its  growth  in  gelatin,  in  milk,  in  bouillon,  and  on  potatoes. 

54.  What  effect  has  it  on  various  sugars,  such  as  maltose,  glucose,  lactose? 

55.  What  are  its  characteristics  as  to  gas  and  indol  production? 

56.  Give  the  differences  in  the  cultural  properties  of  the  colon,  the  hog  cholera, 
and  the  typhoid  bacillus. 

57.  What  are  the  other  names  under  which  white  scours  in  calves  is  known? 

58.  What  is  probably  the  cause  of  this  disease? 

59.  Describe  the  pathologic  lesions  in  (a)  a  very  rapid  case;  (6)  a  less  rapid 


60.  What  other  bacteria  may  be  the  cause  of  diarrhea  in  calves? 

61.  What  is  malignant  catarrh  in  cattle?   What  is  the  organism  that  Leclainche 
claims  is  its  cause? 

62.  What  is  psittacosis? 

63.  What  microorganism  causes  it? 

64.  Is  man  susceptible  to  this  disease?  «. 

65.  Describe  the  Bacillus  typhi  murium.    What  is  Danysz's  bacillus? 

66.  What  are  the  principal  symptoms  and  the  chief  pathologic  lesions  of 
white  diarrhea  in  chicks? 

67.  What,  according  to  Rettger  and  Harvey,  is  the  cause  of  the  disease? 

68.  Describe  the  morphology  of  the  Bacterium  pullorum. 

69.  How  can  it  best  be  obtained  from  animals  dead  from  the  disease? 

70.  Describe  its  cultural  properties. 

71.  What  effect  has  the  Bacterium  pullorum  when  fed  to  or  inoculated  into 
very  young  chicks? 

72.  What  is  the  relation  of  Coccidium  tenellum  to  white  diarrhea  of  chicks? 


CHAPTER    XXV. 

BACILLUS  OF  SWINE  ERYSIPELAS— BACILLUS  OF  MOUSE 
SEPTICEMIA. 

BACILLUS  OF  SWINE  ERYSIPELAS. 

Occurrence  and  Historical. — The  disease  swine  erysipelas,  red  fever 
of  swine;  "Backsteinblattern,"  "Stabchenrothlauf"  (German);  "rouget 
du  pore"  (French),  is  an  acute  septicemic  disease  of  swine  occurring 
either  sporadically  or  epizootically.  The  disease  is  comparatively 
prevalent  in  European  countries.  In  Germany,  89,087  cases,  with  a 
mortality  of  over  80  per  cent.,  were  reported  in  1903;  in  France  the 
annual  loss  is  estimated  at  100,000  animals  and  over,  and  the  disease 
is  also  relatively  prevalent  in  other  countries  of  Europe.  The  affection 
has  been  known  for  a  long  time,  but  prior  to  the  investigations  of 
Pasteur  and  Thuillier,  who,  however,  did  not  discover  its  true  cause, 
it  was  mistaken  for  anthrax  in  swine.  The  true  cause  of  the  disease 
was  found  in  1885  by  Loeffler  and  a  little  later  also  by  Schiitz  in 
a  bacillus  known  as  the  Bacillus  erysipelatis  suis  or  the  bacillus  of 
swine  erysipelas. 

Pathologic  Lesions. — In  the  acute  form  of  the  disease  the  post- 
mortem findings  are  not  very  characteristic.  The  mucosa  of  the 
stomach,  particularly  in  the  neighborhood  of  the  pylorus,  is  inflamed, 
swollen,  and  red,  covered  with  a  tenacious  mucus,  upon  the  removal 
of  which  numerous  hemorrhagic  spots  are  encountered.  The  mucosa 
of  the  small  intestines  is  likewise  inflamed  and  congested,  Peyer's 
patches  are  swollen,  and  here  and  there  superficial  ulceration  is  seen. 
The  latter  also  occurs  in  the  large  intestines,  particularly  in  the  region 
of  the  ileocecal  valve.  The  spleen  is  congested  and  often  somewhat 
enlarged,  and  the  kidneys  show  cloudy  swelling.  In  the  cortical 
substance  of  the  latter,  reddish  points  are  seen.  These  are  the 
inflamed  and  hemorrhagic  glomeruli  (glomerulonephritis).  The  lungs 
are  hyperemic  and  edematous.  The  lymphatic  glands  are  edematous, 
hyperemic,  and  much  swollen.  The  serous  membranes  sometimes 
show  a  fine  fibrinous  deposit,  and  frequently  hemorrhagic  spots.  The 
changes  in  the  skin  from  which  the  disease  has  received  its  name 
consist  in  hemorrhagic  spots,  which  are  due  to  the  great  congestion  of 
superficial  bloodvessels  with  some  blood  extravasation  into  the  sub- 
cutaneous connective  tissue. 

In  the  chronic  form  of  the  disease  an  inflammation  of  the  serous 


CULTURAL  PROPERTIES  299 

lining  of  the  interior  of  the  heart  is  frequently  found.  The  valves 
are  covered  with  fibrinous  and  hemorrhagic  wart-like  deposits,  and 
scattered  ulcerations  are  seen.  This  is  a  pathologic  change  known 
as  endocarditis  verrucosa  and  ulcerosa.  The  specific  bacillus  causing 
the  disease  occurs  in  moderate  numbers  in  the  blood  and  in  larger 
numbers  in  the  spleen,  liver,  and  kidneys,  and  the  verrucous  deposits 
in  the  heart. 

Morphology  and  Staining  Properties. — The  Bacillus  rhusiopathise  suis, 
or  bacillus  of  swine  erysipelas,  can  be  best  seen  in  preparations  made 
from  the  juice  of  the  spleen,  liver,  or  kidneys  of  swine  just  dead  from 
the  disease  or  killed  during  its  last  stages.  It  is  a  very  small,  slender 
bacillus,  only  1  to  1.5  micra  long;  it  occurs  singly  and  in  small 
groups,  sometimes  in  chains  which  are  wavy  or  angular  in  outline. 
Such  chains  are  particularly  clearly  seen  in  the  endocardial  verrucous 
deposits,  which  may  contain  the  organisms  in  very  large  numbers. 
The  bacillus  stains  with  the  ordinary  watery  anilin  stains,  and  is  Gram 
positive.  The  latter  stain  shows  the  bacilli  particularly  well  in  blood 
smears,  or  smears  from  the  organs  mentioned,  in  which  it  is  found 
in  larger  numbers  or  in  sections  of  tissues.  The  bacillus  is  not  motile, 
possesses  no  flagella,  and  forms  no  spores. 

Cultural  Properties. — The  organism  grows  well  at  room  and  at 
incubator  temperatures.  It  grows  well  in  a  very  characteristic  manner 
in  gelatin  as  first  described  by  Loeffler,  Schiitz,  and  Schottelius.  On 
gelatin  plate  cultures  inoculated  from  spleen  juice  or  blood,  hazy, 
bluish-gray,  racemose,  cloudy  spots  appear  on  the  second  or  third 
day;  they  are  situated  a  little  below  the  surface  of  the  medium,  and 
can  only  be  seen  with  some  difficulty  if  the  plate  is  placed  on  a  dark 
background.  If  stab  cultures  in  gelatin  are  made  from  one  of  these 
colonies,  small  round  colonies,  with  lines  radiating  toward  the 
periphery,  appear  along  the  stab;  these  lines  become  divided  and 
finally  form  a  hairy  or  cloudy  mass.  After  six  to  ten  days  the  gelatin 
culture  has  a  very  characteristic  appearance  which  has  been  likened 
to  that  of  a  test-tube  brush.  The  surface  of  the  gelatin  remains  free; 
no  growth  occurs  on  it.  On  a  streak  culture  on  gelatin  slants  the 
colonies  are  more  like  those  on  gelatin  plates.  Sometimes,  however, 
the  colonies  form  round  whitish  or  yellowish-brown  globular  masses, 
without  the  appearance  of  brush-like  extensions.  On  agar  and  blood 
serum  the  bacillus  grows  very  scantily,  but  better  when  the  tubes 
are  kept  in  an  atmosphere  from  which  the  oxygen  has  been  removed 
by  Buchner's  pyrogallic  acid  method.  The  organism  grows  in 
bouillon,  and  first  slightly  clouds  the  medium;  later  a  scanty  grayish- 
white  deposit  is  formed.  It  does  not  grow  on  potatoes  kept  aero- 
bically,  but  it  has  occasionally  been  raised  on  potatoes  kept  in  an 
oxygen-free  atmosphere.  Smears  from  artificial  cultures  show  the 
organism  singly,  in  pairs,  and  in  short,  wavy,  or  angular  chains; 
involution  forms  are  frequently  formed. 


300  BACILLUS  OF  SWINE  ERYSIPELAS 

Resistance. — The  bacillus  of  swine  erysipelas  is  more  resistant 
than  most  pathogenic  non-spore-forming  bacteria.  It  can  remain 
alive  for  one  month  dried  out  and  kept  in  the  incubator  at  37°  C. ;  in 
the  dried  condition  it  resists  sunlight  for  twelve  days ;  and  sometimes, 
in  moist  condition,  a  temperature  of  70°  C.  for  fifteen  minutes. 
According  to  Petri  it  has  survived  in  infected  pork  after  broiling  it 
for  two  and  one-half  hours;  boiling  of  the  meat  in  water,  however, 
promptly  kills  the  bacillus,  which  can  remain  alive  a  long  time  in 
salted  or  smoked  meat  and  in  buried  cadavers.1 

Natural  Infection. — Hogs  are  the  only  animals  subject  to  natural 
infection  with  the  bacillus  of  swine  erysipelas.  They  are  especially 
susceptible  when  they  are  from  three  to  twelve  months  old;  sucking 
pigs  are  not  very  susceptible,  nor  are  animals  older  than  one  year. 
The  latter  have  probably  acquired  immunity  by  a  previous  attack, 
which  may  have  been  so  mild  as  to  have  escaped  attention.  Natural 
infection  occurs  through  the  skin  or  the  intestines;  infection  through 
the  lungs  by  inhalation  has  never  been  proved.  It  is  believed  that 
the  disease  is  most  commonly  contracted  through  the  intestines, 
because  large  numbers  of  bacilli  are  found  in  them  and  also  because 
the  affection  often  occurs  extensively  among  animals  kept  in  one 
place  under  the  same  conditions.  Food  and  water  contaminated  with 
the  feces  of  sick  animals  are  a  fruitful  source  of  spreading  the  disease 
to  large  numbers  of  animals.  The  importation  of  the  disease  into 
previously  uninfected  territories  takes  place  through  sick  animals  or 
their  products.  Both  Cornevin  and  Kitt  have  demonstrated  the  in- 
fective character  of  the  feces  of  sick  animals.  Olt,  Jensen,  and 
Baumeister  have  shown  the  presence  of  erysipelas  bacilli  in  the 
tonsils,  intestines,  and  lymph  glands  of  otherwise  healthy  hogs. 

Artificial  Inoculation. — Gray  and  white  mice,  pigeons,  and  rabbits 
are  very  susceptible  to  artificial  inoculation  with  fresh  cultures  of  the 
bacillus  or  with  blood,  juice  from  the  organs,  etc.,  of  hogs  sick  with 
the  disease  or  dead  from  it.  If  a  mouse  is  inoculated  subcutaneously 
with  a  small  amount  of  infected  material  it  becomes  very  sick  after 
twenty-four  hours,  and  generally  dies  within  four  days  under  repeated 
attacks  of  suffocation.  This  form  of  death  is  quite  characteristic 
in  mice  infected  with  the  bacillus  of  swine  erysipelas.  Field  mice  are 
not  susceptible  to  the  organism.  Pigeons  die  on  the  third  or  fourth 
day  after  inoculation,  and  they  likewise,  though  not  to  so  marked  a 
degree  as  mice,  show  respiratory  difficulty  before  the  fatal  termination. 
If  rabbits  are  inoculated  cutaneously  or  subcutaneously  at  the  ear, 
an  erysipelas-like  swelling  and  redness  appears  at  the  point  of  in- 
jection. The  local  process  may  disappear,  or  it  may  spread  and  lead 
to  a  general  infection  and  death.  Sometimes  death  does  not  occur 
until  after  a  prolonged  cachectic  condition.  If  rabbits  are  inoculated 

1  In  cadavers  it  has  been  found  alive  after  280  days. 


PROTECTIVE  INOCULATIONS  301 

intravenously  they  promptly  die  within  three  to  six  days  from  a 
general  infection.  Inoculation  experiments  on  hogs  or  feeding  of 
infected  material,  as  shown  by  Kitt,  Schiitz,  Schottelius,  and  Preisz, 
lead  to  the  development  of  a  typical  attack  of  erysipelas  with  septi- 
cemia. 

Protective  Inoculations. — The  first  successful  attempt  in  protecting 
experimental  laboratory  animals  and  hogs  against  erysipelas  infection 
were  made  by  Emmerich  and  Mastbaum;  these  were  followed  by 
Pasteur's  protective  inoculations  with  cultures  of  the  bacillus  atten- 
uated for  hogs  by  being  repeatedly  passed  through  rabbits.  The 
modern  methods  now  universally  used  were  worked  out  almost 
simultaneously  by  Leclainche,  Voges  and  Schiitz,  and  Lorenz.  These 
methods  consist  in  injecting  an  immune  serum  of  high  value  and  a 
virulent  culture  of  the  bacillus  of  swine  erysipelas,  either  mixed  or 
simultaneously,  in  different  places  or  at  different  times,  i.  e.,  first  the 
immune  serum,  then  the  culture.  The  immune  serum  is  generally 
prepared  in  the  horse,  occasionally  in  cattle.  Horses  in  order  to 
furnish  an  immune  serum  of  high  value  generally  receive  first  intra- 
venously 100  c.c.  of  a  bouillon  culture  of  virulent  erysipelas  bacilli. 
It  is  necessary  to  begin  with  a  comparatively  large  dose  because 
equines  do  not  react  to  small  doses  of  5  to  50  c.c.  of  bouillon  cultures. 
The  doses  are  increased  in  intervals  of  eight  to  ten  days  to  150,  200, 
250,  300,  and  even  to  500  c.c.  In  this  manner  a  serum  of  high  value 
is  generally  obtained  in  about  two  months.  When  the  reaction  after 
the  last  injection  has  subsided  the  blood  is  drawn,  collected,  and 
treated  in  the  usual  aseptic  manner  and  prepared  for  preservation  by 
the  addition  of  0.5  per  cent,  carbolic  acid.  Kitt,  Schubert,  and  Pretter 
have  also  prepared  an  antiserum  in  a  similar  manner  in  cattle.  It  is 
claimed  by  some  that  a  mixture  of  horse  and  cattle  serum,  a  so-called 
"double  serum,"  is  more  powerful  in  its  effect  than  the  horse  serum 
alone,  but  this  is  denied  by  others.  The  antiserum  must  be  tested 
before  use.  The  best  animals  for  this  purpose  are  gray  house  mice. 
A  good  strong  immune  serum  should,  in  a  dose  of  0.01  gram,  protect 
a  mouse  of  15  grams  against  0.1  gram  of  virulent  bouillon  culture 
of  the  erysipelas  bacillus.  An  immune  erysipelas  serum  from  horses 
or  cattle  meeting  with  these  requirements  is  used  in  practice  as  follows: 

For  Therapeutic  Purposes  in  Hogs  Sick  with  Erysipelas. — A  hog 
weighing  up  to  100  pounds  receives  10  c.c.;  hogs  from  100  to  250 
pounds,  20  c.c.;  hogs  weighing  over  250  pounds,  30  c.c.  The  in- 
jection may  be  made  at  any  place  of  the  body,  but  the  base  of  the 
external  ear,  or,  if  larger  amounts  are  used,  the  knee  fold  are  generally 
preferred.  The  therapeutic  value  of  the  injection  is  the  better  the 
earlier  in  the  course  of  the  disease  it  is  used. 

For  Protective  Purposes  by  the  Simultaneous  Method. — As  already 
stated,  protective  inoculation  may  be  made  by  the  simultaneous 
method,  or  with  an  interval  between  the  serum  and  the  culture 


302  BACILLUS  OF  SWINE  ERYSIPELAS 

injection.     The  doses  recommended  for  hogs  of  different  sizes  are 
the  following: 

For  hogs  up  to  50  pounds 3  c.c.  antiserum 

For  hogs  from  50  to  100  pounds 5  c.c.  antiserum 

For  hogs  from  100  to  250  pounds       . 8  c.c.  antiserum 

For  hogs  from  150  to  200  pounds 10  c.c.  antiserum 

For  hogs  over  300  pounds 15  c.c.  antiserum 

The  bouillon  culture  employed  for  injection  must  be  young  and 
virulent.  It  is  used  in  doses  of  from  0.25  to  1  c.c.,  according  to  the 
weight  of  the  animal,  but  between  these  limits  very  great  accuracy 
in  the  dose  is  unnecessary.  In  the  simultaneous  method  the  immune 
serum  and  the  culture  may  be  mixed,  or  they  may  be  injected  at  the 
same  time  in  two  different  spots.  When  the  serum  is  first  injected 
alone  the  culture  must  be  injected  after  three  to  five  days.  The 
passive  immunization  by  the  serum  alone  produces  immunity  for  a 
very  short  period  only,  but  the  combined  passive  and  active  immu- 
nization protects  the  animals  treated  for  a  period  variously  estimated 
at  from  six  to  twelve  months.  The  statistics  from  France,  Germany, 
and  Austria  concerning  the  value  of  the  combined  protective  inocu- 
lation of  hogs  against  erysipelas  are  very  favorable. 

Transmission  to  Man. — Preisz  and  other  authors  have  reported  a 
few  cases  of  persons  who  having  handled  hogs  sick  with  erysipelas 
or  their  meat,  contracted  a  slight  erysipeloid  lesion  of  the  skin,  transi- 
tory in  character,  and  not  leading  to  any  grave  general  disturbances. 


THE  BACILLUS  OF  MOUSE  SEPTICEMIA. 


Koch,  when  studying  wound  infection  diseases  early  in  his  career, 
discovered  a  very  minute  bacillus,  very  fatal  to  mice,  which  he  called 
the  Bacillus  murisepticus,  or  the  bacillus  of  mouse  septicemia.  After 
Loeffler  had  discovered  the  bacillus  of  swine  erysipelas  it  was  found 
that  the  latter  and  the  bacillus  of  mouse  septicemia  were  so  similar  in 
morphologic  and  cultural  properties  that  it  appeared  reasonable  to 
consider  them  as  one  species.  The  question,  however,  is  not  yet  fully 
settled.  It  is  discussed  by  Preisz,  who  has  given  considerable  attention 
to  this  matter  in  his  article  on  swine  erysipelas.1  Preisz's  statements 
are  to  the  following  effect.  The  question  whether  anybody  has 
succeeded  in  finding  the  bacillus  of  hog  erysipelas  in  the  outside 
world  is  intimately  connected  with  the  question  whether  the  bacillus 
is  identical  with  that  of  mouse  septicemia.  Koch  found  the  bacillus 
in  putrefying  fluids,  Loeffler  observed  an  epidemic  among  his  mice 
due  to  it,  Johne  cultivated  it  from  putrid  meat,  and  Preisz  from 
putrid  cattle  blood. 

The  morphology  and  cultural  properties  of  the  Bacillus  murisep- 

1  Kolle  and  Wassermann's  Manual,  vol.  iii. 


QUESTIONS  303 

ticus  and  of  the  bacillus  of  swine  erysipelas  may,  under  certain  cir- 
cumstances, show  the  very  greatest  similarity,  but  they  may  also,  under 
absolutely  like  circumstances,  show  more  or  less  apparent  differences. 
The  mouse  bacillus  may  appear  in  the  blood  of  infected  animals  still 
finer  than  the  erysipelas  bacillus.  They  may  both  show  marked 
differences  as  to  cultural  properties  in  gelatin  stick  cultures.  The 
mouse  bacillus  may  grow  very  much  more  rapidly  than  the  erysipelas 
bacillus  under  absolutely  the  same  conditions.  These  differences, 
however,  do  not  necessarily  mean  that  the  organisms  are  two  species. 
No  investigator  has  yet  succeeded  in  producing  a  typical  erysipelas 
in  swine  by  inoculation  with  the  Bacillus  murisepticus,  but  Luepke 
claims  to  have  produced  the  mild  form  of  swine  erysipelas  known 
in  German  as  "Backsteinblattern"  (brick-pox).  Rabbits  and  hogs, 
according  to  Lorenz,  can  be  immunized  against  the  bacillus  of 
erysipelas  by  inoculation  with  the  Bacillus  murisepticus. 

From  the  above  it  appears  that  the  question  of  the  identity  or  non- 
identity  of  these  two  bacilli  cannot  yet  be  considered  as  satisfactorily 
settled. 

QUESTIONS. 

1.  What  are  the  other  names  for  swine  erysipelas? 

2.  What  kind  of  a  disease  is  it? 

3.  What  is  the  cause  of  the  disease?    Who  discovered  it? 

4.  Describe  the  pathologic  lesion  in  the  acute  form  of  the  disease. 

5.  What  is  a  glomerulonephritis? 

6.  What  is  an  endocarditis  verrucosa?    Is  it  found  in  swine  erysipelas? 

7.  What  animals  are  susceptible  to  natural  infection?      How  is  the  disease 
contracted? 

8.  Is  the  bacillus  of  swine  erysipelas  sometimes  found  in  healthy  hogs,  and 
where? 

9.  What  laboratory  animals  are  susceptible  to  artificial  infection? 

10.  Under  what  symptoms  does  a  mouse  die  when  inoculated  with  material 
containing  the  bacillus  of  swine  erysipelas? 

11.  How  do  rabbits  act  after  subcutaneous  and  after  intravenous  injection? 

12.  Describe  the  morphology  and  staining  properties  of  the  Bacillus  rhusio- 
pathwe  suis. 

13.  Describe  the  cultural  properties,  particularly  the  appearance  of  a  gelatin 
stick  culture. 

14.  Discuss  the  resistance  of  the  bacillus. 

15.  Who  developed  the  modern  methods  of  serum  therapy  against  swine 
erysipelas? 

16.  How  is  the  antiserum  prepared  from  horses  or  cattle? 

17.  What  is  meant  by  a  double  erysipelas  serum? 

18.  How  is  the  serum  tested  as  to  its  protective  power? 

19.  In  what  doses  is  the  immune  serum  used  when  it  is  employed  as  a  curative? 

20.  How  is  protective  inoculation  practised?     Describe  the  three  different 
methods. 

21.  What  kind  of  cultures  are  used  in  active  immunization  and  in  what  doses? 

22.  What  is  the  bacillus  of  mouse  septicemia?    Who  discovered  it? 

23.  What  is  the  relation  of  the  bacillus  of  mouse  septicemia  to  that  of  swine 
erysipelas? 

24.  Is  swine  erysipelas  ever  transmitted  to  man  ? 


CHAPTEE    XXVI. 

GLANDERS  BACILLUS. 

Occurrence  and  Historical. — Glanders,  malleus,  malleosis,  farcy, 
"Rotz"  (German),  "morve"  (French),  is  a  disease  which  has  been 
known  to  mankind  for  a  long  time.  The  name  glanders  refers  to  the 
lymph-gland-like  swelling  and  to  the  swelling  of  the  lymph  glands. 
The  designation  malleus,  malleosis,  morve,  is  derived  from  a  Greek 
word  meaning  a  bad  disease,  and  the  German  word  "Rotz"  is  a 
generally  employed  but  rather  vulgar  designation  for  a  mucopurulent 
discharge  from  the  nose.  It  is  claimed  that  the  word  malleus  was 
introduced  by  the  celebrated  Greek  philosopher  and  naturalist, 
Aristo teles.  The  contagious  nature  of  the  disease  among  horses  was 
recognized  as  early  as  the  fourth  century  after  Christ,  but  this  fact 
was  afterward  forgotten  and  not  rediscovered  until  about  200  years 
ago. 

This  infectious,  contagious  disease  is  found  particularly  among 
equines,  but  it  is  also  communicable  to  man,  sheep,  goats,  camels, 
carnivora,  etc.  Laboratory  animals,  like  guinea-pigs,  rabbits,  etc.,  are 
likewise  very  susceptible.  It  is  caused  by  a  microorganism  known 
as  the  Bacillus  mallei,  first  discovered  in  1882  by  Loeffler  and  Schiitz. 
Cattle  are  absolutely  immune  to  the  Bacillus  mallei;  pigs  are  very 
slightly  susceptible  and  can  be  infected  only  with  difficulty.  Domestic 
birds  are  likewise  immune. 

Glanders  is  very  prevalent  almost  over  the  entire  world.  It  is 
found  in  Europe,  Asia,  Africa,  and  America.  It  has  been  introduced 
into  the  Philippine  Islands,  but  so  far  it  has  been  excluded  from 
Australia,  including  Tasmania  and  New  Zealand. 

Mode  of  Infection  and  Pathologic  Changes. — The  disease  may  take 
either  an  acute,  a  subacute,  or  a  very  chronic  course,  the  latter  extend- 
ing over  years,  and  in  each  case  it  leads  to  local  inflammatory  changes, 
with  the  formation  of  granulation  tissue.  The  glanders  bacillus 
frequently  gains  entrance  through  the  upper  respiratory  passages, 
and  first  becomes  localized  in  the  nasal  mucosa;  but  it  also  frequently 
enters  through  wounds  and  abrasions  of  the  skin  or  epithelial  covering. 
From  the  portal  of  entrance  the  glanders  bacillus  spreads  along  the 
lymphatics,  w^here  it  multiplies;  later  it  invades  the  lymph  glands  and 
finally  the  internal  organs.  The  infection  is  spread  from  animal  to 
animal  by  direct  contact  or  by  coughing,  in  consequence  of  which 
small  expelled  particles  of  morbid  material  coming  from  a  diseased 
animal  are  inhaled  by  a  healthy  one.  The  infection  is  spread  indirectly 


MODE  OF  INFECTION  AND  PATHOLOGIC  CHANGES       305 

through  feed,  bedding,  harness,  etc.,  which  have  been  soiled  with 
the  discharge  from  glanderous  ulcerations  or  suppurating  foci. 

Wherever,  in  natural  infection,  glanders  bacilli  have  gained  entrance 
into  the  body  of  a  susceptible  animal,  they  multiply  locally  and 
cause  a  cell  necrosis.  This  is  followed  by  an  inflammatory  reaction 
with  hyperemia,  transudation,  and  emigration  of  leukocytes.  The 
inflammatory  reaction,  however,  does  not  limit  the  infection  but  the 
bacilli  continue  to  multiply  and  the  necrotic  processes  spread  and 
with  them  the  inflammatory  reaction.  The  infection  is  spread  both 
by  direct  extension  from  the  multiplying  glanders  bacilli  and  by 
transportation  of  bacilli  to  neighboring  or  more  distant  parts  of  the 
body  through  the  agency  of  leukocytes  which  have  taken  them  up 
but  have  not  killed  them.  At  these  points  they  again  multiply  and 
give  rise  to  the  same  lesions  which  they  produced  at  the  original 
place  of  entrance.  The  infection  may  also  be  spread  in  a  glanderous 
animal  by  the  discharge  from  the  lesions  flowing  over  a  mucous 
membrane.  In  this  manner  the  disease  may  spread  from  the  nose  to 
the  trachea,  bronchi,  and  lungs;  or,  in  primary  pulmonary  cases,  the 
mode  of  extension  may  be  from  the  lungs  outward. 

The  anatomical  changes  which  glanders  produces  in  the  skin, 
mucous  membranes,  and  various  internal  organs  vary  according  to 
the  location  and  the  virulency  and  acuteness  or  chronicity  of  the 
process.  According  to  Kitt,  four  anatomical  types  of  lesions  are 
distinguished,  namely:  The  glanders  nodule,  the  glanders  abscess, 
and  ulceration,  the  glanders  induration,  and  the  glanders  infiltration. 

Nodules. — The  formation  of  the  glanders  nodule  may  be  studied 
in  the  nose  of  the  horse.  Following  the  invasion  and  multiplication 
of  the  glanders  bacillus  a  small,  slightly  elevated,  grayish- white,  trans- 
parent nodule,  varying  in  size  from  a  small  shot  to  a  pea,  is  formed 
as  the  result  of  the  inflammatory  cellular  infiltration.  In  consequence 
of  the  necrosis  in  the  interior  of  the  nodule  a  small  abscess  cavity 
in  its  centre  soon  develops.  When  the  necrosis  extends  outward  far 
enough  a  loss  of  substance  occurs  at  the  highest  point  and  an  open 
ulcer  is  formed. 

Ulcers. — The  glanders  ulcers  are  at  first  generally  spherical,  but 
they  become  irregular,  either  by  an  irregular  extension  of  the  necrosis 
at  the  periphery  or  by  the  confluence  of  neighboring  spreading  ulcers. 
The  ulceration  in  glanders  have  an  excavated,  eaten-out  appearance. 
They  are  surrounded  by  elevated  margins  and  the  ulcer  is  covered 
by  a  thin,  greenish-white,  seropurulent  discharge,  sometimes  slightly 
stained  with  blood.  If  dried  out,  it  covers  the  ulcer  as  a  dirty  grayish- 
brown  crust.  In  the  skin  the  glanders  nodules  may  be  as  large  or 
larger  than  a  hazel  nut  and  a  distinctly  palpable  abscess  may  be  felt 
before  the  outer  portion  of  the  abscess  wall  is  broken  through  and  an 
ulcer  formed.  When  glanders  nodules  are  first  formed  in  the  lungs, 
as  in  the  pulmonary  form  of  the  disease,  they  are  small,  grayish-white, 
translucent  nodules  which  somewhat  resemble  young  tubercles.  The 
20 


306  GLANDERS  BACILLUS 

bronchial  glands  become  studded  with  such  nodules  and  later  with 
abscesses.  In  the  skin  the  glanders  abscesses,  before  leading  to 
external  ulceration,  contain  a  thin,  greasy,  yellowish  or  yellowish-red 
pus,  occasionally  mixed  with  a  necrotic  cell  detritus. 

Indurations. — The  glanders  indurations  are  frequently  found  in  the 
nose,  where  they  present  themselves  in  two  types.  In  the  one  there  is 
a  cicatricial  formation,  following  an  ulcerative  process  which  has 
come  more  or  less  to  a  standstill;  in  the  other  in  which  the  glanders 
virus  may  not  have  been  of  a  very  virulent  type  from  the  beginning, 
and  may  not  have  led  to  ulceration,  there  is  instead  little  necrosis 
and  much  connective-tissue  proliferation,  with  the  formation  of 
fibrous  connective  tissue.  Bands  and  ridges,  stellate  and  radiating 
masses,  projecting  slightly  over  the  surface  of  the  surrounding  more 
healthy  mucosa  are  formed,  either  secondarily  after  ulceration  or 
primarily.  Sometimes  the  masses,  instead  of  being  rather  firm,  have 
a  softer  character,  and  the  purplish  color  of  young  granulation  tissue. 

Infiltrations. — The  glanders  infiltrations  are  found  particularly  in 
the  lungs  in  the  pulmonary  type  of  the  disease.  In  the  horse  this 
type  assumes  first  the  character  of  a  bronchopneumonia.  Small 
areas  of  consolidation  with  nodule  formation  appear.  These  areas 
are  of  an  elastic  consistency  and  a  gelatinous  yellowish  color,  some- 
times mixed  with  red  or  light  brown.  As  they  increase  in  age  these 
areas  spread  and  become  confluent,  forming  larger  sarcoma-like 
masses,  which  when  incised  are  either  homogeneous  and  grayish  red 
or  white  in  color,  or  they  may  be  honeycombed  with  smaller  or  larger 
abscess  cavities,  filled  with  pus  and  a  cheesy  or  moist  chalk-like  gran- 
ular material.  Associated  with  this  condition  in  the  lungs  is  an 
enlargement  and  hyperplastic  inflammation  and  purulent  cavity 
formation  in  the  thoracic  lymph  glands.  A  diffuse,  gelatinous 
infiltration  is  formed  in  the  lymph  channels.  Such  infiltrations  of 
a  gelatinous  character  are  also  frequently  found  in  the  lymph  channels 
of  the  skin  in  skin  glanders. 

Pulmonary  Glanders. — Pulmonary  glanders  in  the  horse,  as  already 
stated,  generally  first  assumes  the  type  of  a  lobular  or  broncho- 
pneumonia,  no  matter  what  the  sequel  may  be.  In  the  cat  tribe  it 
generally  begins  with  the  consolidation  of  an  entire  or  of  several 
lobes,  and  has  anatomically  the  character  of  a  lobar,  fibrinous,  or 
croupous  pneumonia.  According  to  McFadyean  the  lungs  always 
sooner  or  later  become  involved  in  glanders,  wherever  the  original 
portal  of  entrance  may  have  been. 

Microscopic  Changes. — These  vary  somewhat  in  the  acute  and 
chronic  type.  In  the  acute  type  a  central  area  of  marked  necrosis 
with  cell  degeneration  and  nuclear  fragmentation  is  generally  found. 
Around  the  necrotic  area  many  polynuclear  leukocytes  are  seen;  many 
of  them  are  themselves  degenerated,  others  intact.  The  vessels  in 
the  inflamed  but  not  yet  necrotic  area  are  greatly  congested.  In  the 
pulmonary  cases  in  the  horse,  multinuclear  giant  cells  are  frequently 


MODE  OF  INFECTION  AND  PATHOLOGIC  CHANGES       307 

seen  in  the  inflammatory  zone  next  to  the  necrotic  area.  In  the  slow 
chronic  cases  the  histologic  examination  shows  the  presence  of  much 
fibrous  connective  tissue. 

Susceptibility. — That  man  is  not  as  susceptible  to  the  natural 
glanders  infection  as  the  solidipeds,  can  be  seen  in  the  fact  that  many 
men  handle  affected  horses,  and  yet,  on  the  whole,  the  number  of 
persons  contracting  the  disease  is  comparatively  small.  On  the 
other  hand,  the  number  of  scientific  investigators  working  with  the 
Bacillus  mallei  in  the  laboratory  and  contracting  a  fatal  infection  has 
been  alarmingly  large.  Hence,  students  working  with  live  glanders 

FIG.  147 


The  pustular  eruption  of  acute  glanders  in  man  as  exhibited  on  the  day  of  the  patient's 
death,  twenty-eight  days  after  the  initial  chill.     (Zeit.) 

cultures  should  be  exceedingly  careful.  The  first  infection  of  glanders 
in  man  was  recognized  by  Lorin  in  1812.  By  far  the  greatest  number 
of  cases  of  natural  glanders  infection  in  human  beings  occurs  among 
hostlers,  drivers,  farmers,  horse  butchers,  and  other  habitual  handlers 
of  horses.  In  these  trades  the  disease  is  generally  contracted  through 
abrasions  or  wounds  of  the  skin,  commonly  leading  to  the  formation  of 
a  pustular  eruption  which  has  very  frequently  been  mistaken  for  small- 
pox and  also  for  a  gangrenous  erysipelas.  In  laboratory  workers  the 
disease  generally  begins  as  a  respiratory  (nasal  and  pulmonary)  infec- 
tion. Even  this  mode  of  infection,  however,  is  likely  to  lead  to  a  pustu- 


308  GLANDERS  BACILLUS 

lar,  cutaneous  eruption  before  death  occurs.  In  man  the  various  types 
usually  take  an  acute  rapid  course.  The  chronic  type  of  cutaneous 
glanders  with  the  formation  of  abscesses,  ulcers,  and  lymphatic 
swelling  and  involvement  has  also  been  observed  in  man.  Cases  of 
this  kind  have  formerly  been  mistaken  for  tertiary  syphilitic  lesions. 
More  acute  cases  resembling  smallpox  in  man  have  been  reported  by 
Wherry,  Zeit,  Bevan  and  Hamburger,  and  others. 

Morphology. — The  Bacillus  mallei  varies  considerably  in  size  and 
shape  according  to  the  culture  medium  on  which  it  has  been  grown. 
It  is,  as  a  rule,  rather  slender;  occasionally,  however,  it  is  short  and 
plump.  It  is  from  2  to  5  micra  long  and  from  0.5  to  1  micron  in 
diameter.  On  old  potato  cultures  the  bacillus  sometimes  appears 
in  long  filaments  forming  intertwined,  irregular  masses.  Bacilli  of 
the  ordinary,  most  common  type  are  generally  not  perfectly  straight, 
but  slightly  curved.  They  do  not  stain  uniformly,  but  somewhat  in 
the  manner  of  the  diphtheria  bacillus.  The  Bacillus  mallei  is  not 
actively  motile,  but  shows  a  very  lively  molecular  movement;  it  does 
not  form  spores  nor  does  it  stain  well  with  the  ordinary  watery  anil  in 
stains,  but  takes  better  the  stronger  stains  such  as  Loeffler's  alkaline 
methylene  blue,  Kuehne's  carbolmethylene  blue  or  carbol-thionin. 
Loeffler  has  recommended  the  following  method  for  staining  for 
glanders  bacilli  in  smears  from  suspected  pus  or  necrotic  material: 

1.  Prepare  the  cover-glass   smear  in  the  usual   manner,  air  dry, 
fix  and  float  the  cover-glass  for  five  minutes  on  Loeffler 's  alkaline 
methylene  blue. 

2.  Dip  for  one  second  into  a  1  per  cent,  watery  solution  of  acetic 
acid  to  which  enough  of  a  watery  solution  of  tropeolin  00  has  been 
added  to  give  it  a  Rhine-wine  yellow  color. 

This  last  step  decolorizes  the  cell  protoplasm  entirely  and  the 
nuclei  partially,  so  that  the  deep  blue  stained  bacilli  can  be  more 
readily  found. 

Cultural  and  Biologic  Properties. — The  Bacillus  mallei  can  be 
obtained  in  pure  culture  without  great  difficulty.  It  is  generally 
impossible  to  obtain  it  directly  from  the  discharges  from  glanderous 
lesions  in  the  horse.  As  a  rule,  it  is  necessary  first  to  inoculate  a  guinea- 
pig  in  the  manner  described  below.  The  bacillus  grows  best  between 
30°  to  40°  C.;  growth  ceases  at  and  below  20°  C.  and  above  43°  C. 
It  is  a  strict  parasite  which  has  never  been  found  under  natural  con- 
ditions except  in  connection  with  cases  of  glanders.  If  cultures  have 
been  raised  for  many  generations  on  artificial  media  the  bacillus 
may  also  grow  at  temperatures  lower  than  20°  C.  The  organism  grows 
much  better  in  artificial  cultures  in  the  presence  of  oxygen;  in  its 
absence  there  is  only  a  very  poor  development.  The  culture  media 
may  be  faintly  alkaline,  neutral  or  faintly  acid;  the  latter  reaction  is 
most  favorable.  The  addition  of  glycerin,  4  or  5  per  cent,  to  the 
agar  or  bouillon,  is  advantageous  to  the  development  of  the  Bacillus 
mallei.  In  ordinary  bouillon  or  glycerin  bouillon  the  Bacillus  mallei 


CULTURAL  AND  BIOLOGIC  PROPERTIES  309 

grows  rapidly,  and  the  medium  after  twenty-four  hours  shows  a  slight 
uniform  clouding;  later  a  whitish,  slimy  sediment  is  formed  without 
clearing  of  the  supernatent  fluid.  If  the  fluid  is  not  disturbed  a 
whitish,  slimy  surface  pellicle  is  also  formed,  and  even  after  this  has 
sunk  to  the  bottom  a  white  ring  or  margin  remains  at  the  circum- 
ference of  the  surface  and  the  glass.  On  slightly  acid  glycerin  agar 
the  organism  develops  well,  but  there  is  nothing  characteristic  about 
the  growth.  The  colonies  are  first  flat,  dull  white  or  grayish,  trans- 
lucent; later  they  become  somewhat  yellowish  or  perhaps  even  some- 
what reddish  yellow.  The  colonies  rapidly  become  confluent  in  the 
incubator,  slowly  at  lower  room  temperatures.  The  organism  grows 
well  on  horses'  and  sheep's  blood  serum,  and  not  so  well  on  cattle 
blood  serum.  The  colonies  after  about  three  days  appear  as  yellowish, 
translucent  drops  on  the  surface  of  the  coagulated  serum.  They 
possess  a  tenacious,  slimy,  viscid  consistency.  After  eight  to  ten  days 
the  growth  on  blood  serum  becomes  opaque  and  grayish  white.  On 
potatoes  the  growth  of  the  Bacillus  mallei  is  most  characteristic;  it 
appears  after  about  two  days  as  a  delicate,  yellowish,  translucent 
cover,  and  on  the  third  day  assumes  an  amber  color.  After  six  to 
eight  days  the  growth  has  become  quite  abundant.  It  is  now  opaque 
and  has  lost  most  of  its  transparency,  being  reddish  yellow  in  color. 
The  surface  of  the  potato  not  covered  by  the  growth  has  assumed 
a  faintly  green  hue.  This  zone,  however,  may  not  be  very  noticeable 
or  it  may  be  yellowish  green  or  brownish  green.  In  using  potatoes 
for  the  cultivation  of  the  Bacillus  mallei  it  is  important  to  select  such 
as  are  not  too  acid,  or,  still  better,  to  correct  the  acidity  before  sterili- 
zation. Potatoes  which  have  been  frozen  or  which  have  begun  to 
germinate  should  not  be  used,  because  they  are  likely  to  contain 
sugar  from  which  the  Bacillus  mallei  forms  acids.  It  is  best  to 
immerse  the  disks  or  pieces  of  potato,  after  washing  and  peeling,  for 
one  hour  in  a  0.5  to  0.7  per  cent,  solution  of  bicarbonate  of  soda  and 
then  to  sterilize  them.  In  this  manner  a  material  of  not  too  high 
acidity  is  obtained.  Certain  other  bacteria  when  growing  on  potato 
form  a  growth  more  or  less  similar  to  that  of  the  Bacillus  mallei.  The 
most  important  of  these  is  the  Bacillus  pyocyaneus.  This  organism 
produces  a  yellowish-brown  growth,  which,  however,  is  not  trans- 
parent; older  colonies  also  exhibit  a  mother-of-pearl  luster  not  shown 
by  the  Bacillus  mallei.  A  simple  test  to  differentiate  between  the 
two  organisms  when  grown  on  potatoes  and  closely  resembling  each 
other  is  to  spread  some  of  the  growth  on  a  piece  of  filter  paper  and 
expose  it  to  ammonia  vapor.  If  the  growth  is  Bacillus  pyocyaneus 
a  characteristic  bluish-green  color  at  once  appears;  this  is  not  shown 
when  the  growth  is  Bacillus  mallei.  An  evidently  rare  pseudomalleus 
bacillus,  discovered  by  Babes,  forms  a  growth  like  the  genuine  glanders 
bacillus  on  potatoes,  and  can  be  accurately  differentiated  only  by 
animal  experiments.  In  guinea-pigs,  field  mice,  and  cats  it  produces 
local  processes  only,  but  kills  rabbits. 


310  GLANDERS  BACILLUS 

Resistance. — The  Bacillus  mallei  may  be  kept  alive  for  a  long  time 
in  artificial  cultures,  provided  these  are  sealed  and  kept  in  a  cool, 
dark  place.  The  bacillus  is  easily  killed  by  heat,  an  exposure  for 
ten  minutes  at  56°  C.  destroys  it.  It  is  likewise  easily  killed  by  the 
ordinary  antiseptics,  such  as  corrosive  sublimate,  carbolic  acid,  etc. 

Bose  reported  that  he  exposed  pieces  of  cotton,  etc.,  infected  with 
young,  virulent  glanders  bacilli  to  the  action  of  formalin  vapors  and 
that  they  were  killed  after  five  hours.  The  author,  however,  has 
found  that  young  cultures  on  glycerin  agar  tubes,  exposed  after  the 
removal  of  the  cotton  plugs  in  a  tightly  closed  anatomical  jar  to 
formalin  vapors,  survived  after  three  days.  It  appears,  accordingly, 
that  formalin  vapors  are  not  to  be  depended  upon  in  disinfecting 
stables  where  cases  of  glanders  have  occurred.  Sulphuric  acid  in  J  to 
2  per  cent,  solutions,  milk  of  lime,  chloride  of  lime,  and  the  chemicals 
of  the  carbolic  acid  group  are  dependable  disinfectants  for  glanders- 
infected  buildings,  harness,  etc. 

Diagnosis. — In  typical  advanced  cases  the  diagnosis  of  glanders  may 
be  made  from  the  clinical  findings.  As  a  rule,  an  accurate  diagnosis 
cannot  be  made  from  the  microscopic  examination  of  a  nasal  or 
cutaneous  purulent  discharge,  but  only  after  inoculating  a  guinea- 
pig  or  after  the  mallein  test  or  the  agglutination  test. 

The  microscopic  examination  of  virulent  discharges  is  much 
invalidated  by  the  fact  that  they  generally  contain  a  mixture  of 
bacteria  among  which  it  is  difficult  and  often  impossible  to  recognize 
the  glanders  bacillus.  The  probability  of  a  successful  microscopic 
diagnosis  is  much  greater  when  the  material  for  examination  consists 
of  soft  necrotic  material  from  the  interior  of  a  not  yet  ulcerated  or 
opened  glanders  nodule,  either  from  the  nasal  mucosa,  the  skin  or  a 
submaxillary  gland.  The  necrotic  or  purulent  material  is  spread  on 
a  cover-glass,  air  dried,  fixed,  and  stained  with  Kiihne's  carbol 
methylene  blue  or  with  Loeffler's  methylene  blue,  and  then  washed, 
as  already  described,  in  an  acetic-acid  tropeolin  00  watery  solution. 
Even  in  favorable  material  glanders  bacilli  are  present  only  in  mod- 
erate numbers,  but  a  diagnosis  may  be  obtained  if  they  are  character- 
istic in  shape  and  staining  properties  (Gram  negative)  and  other 
bacteria  are  absent.  The  attempt  may  also  be  made  to  raise  pure 
cultures  from  such  material  obtained  aseptically  and  showing  only 
one  kind  of  bacillus. 

The  Biologic  Test  for  Glanders  (Strauss'  Test). — The  best  animal 
for  the  inoculation  test  for  glanders  is  the  male  guinea-pig.  The  test 
is  made  as  follows:  Some  of  the  suspected  material  obtained  under 
very  aseptic  precautions  is  rubbed  up  with  sterile  physiologic  salt 
solution  and  1  to  2  c.c.  of  the  mixture  is  injected  with  a  sterile  hypo- 
dermic syringe  into  the  peritoneal  cavity  of  the  animal.  The  injection 
is  made  immediately  above  the  symphysis  pubis.  When  the  material 
contains  virulent  glanders  bacilli  a  swelling  of  the  testicles,  which  are 
now  hot  and  painful,  develops  after  two  to  four  days.  If  the  animal 


THE  MALLEIN  TEST  311 

is  undisturbed  the  testicles  later  ulcerate,  break  through  the  skin,  and 
discharges  a  purulent  fluid  containing  many  bacilli.  The  animal 
becomes  emaciated  and  dies  after  ten  to  twelve  days.  The  post- 
mortem examination  shows  numerous  glanders  abscesses  in  the 
testicles,  the  lymph  nodes  and  in  the  internal  organs,  such  as  the  liver, 
spleen,  kidneys,  lungs,  etc.  When  the  test,  however,  is  made  for 
diagnostic  purposes  the  animal  is  not  allowed  to  die,  but  is  killed  as 
soon  as  the  testicle  shows  a  marked  inflammatory  swelling,  which  is 
generally  the  case  after  two  to  three  days  and  a  postmortem  examination 
is  made.  In  the  case  of  glanders  the  examination  of  the  testicles 
shows  small  grayish-white  transparent  nodules,  sometimes  even 
small  abscesses,  and  smears  from  the  nodules  and  abscesses  show 
the  typical  glanders  bacilli.  If  the  testicles  are  opened  under  aseptic 
precautions  the  glanders  nodule  can  also  be  used  to  obtain  pure 
cultures  of  the  Bacillus  mallei.  It  is  always  necessary  to  examine 
smears  from  the  swollen,  inflamed  testicles  of  the  guinea-pig  micro- 
scopically and  to  stain  with  Loeffler's  or  with  Kiihne's  stains  and  by 
Gram's  method,  because  certain  other  bacteria  also  bring  about  an 
orchitis  (inflammation  of  the  testicles)  if  inoculated  intraperitoneally 
into  a  male  guinea-pig.  These  bacteria,  however,  are  all  Gram 
positive.  They  are  Nocard's  bacillus  of  ulcerative  lymphangitis  of 
the  horse.  Preisz's  bacillus  of  pseudotuberculosis  of  the  sheep, 
Kutscher's  bacillus  found  once  in  the  nasal  secretion  of  a  horse,  and 
also  sometimes  the  Bacillus  pyocyaneus.  Young  cats  may  also  be 
used  for  subcutaneous  inoculation  with  glanders-suspected  material. 
They  develop  first  a  local  swelling  and  ulceration  and  later  a  general 
glanders  infection. 

The  Mallein  Test. — Mallein1  is  a  vaccine,  prepared  from  killed, 
virulent  glanders  bacilli.  It  is  injected  into  a  soliped  suspected  of 
glanders,  and  if  the  disease  is  present  a  typical  local  and  general 
reaction  occurs.  Very  little  is  known  concerning  the  mode  of  action 
of  the  Bacillus  mallei,  but  there  is  good  reason  to  believe  that  its 
pathogenic  properties  depend,  if  not  exclusively,  at  least  largely,  upon 
resistant  endotoxins.  The  latter  are  present  in  the  mallein;  they 
represent  the  antigen  (see  chapter  on  Antibodies),  which  gives  rise  to 
the  formation  of  specific  glanders  antibodies.  The  local  and  general 
reaction  in  malleus-infected  horses,  when  injected  with  mallein,  is 
probably  due  to  an  interaction  between  the  malleus  antigen  and  the 
malleus  antibodies. 

Mallein  is  prepared  according  to  various  methods;  that  of  Roux, 
of  the  Paris  Pasteur  Institute,  is  as  follows:  The  virulent  cultures  of 
glanders  bacilli  are  obtained  by  long-continued  intravenous  injections 
of  bacilli  into  rabbits.  When  the  cultures  have  become  so  virulent 
that  they  will  kill  rabbits  in  thirty  hours  they  are  grown  in  flasks 

The  term  mallein  was  formed  on  the  analogy  of  the  word  tuberculin.  The  term  was 
first  used  by  Helman,  the  original  maker  of  the  vaccine.  German  writers  sometimes  use 
the  term  "Rotzlymphe,"  and  French  authors  the  word  "morvin,"  for  mallein. 


312  GLANDERS  BACILLUS 

containing  250  c.c.  glycerin  bouillon.  After  one  month's  incubation 
at  35°  C.  these  cultures  are  sterilized  in  the  autoclave  under  atmos- 
pheric pressure  at  100°  to  115°  C.  They  are  then  evaporated  down 
on  a  water  bath  to  one-tenth  of  their  original  volume  and  filtered 
through  a  particular  filter  paper.  The  concentrated  end-product 
of  these  procedures  is  known  as  the  raw  mallein,  or  "mallein  brute." 
It  is  a  dark  brown,  syrupy  fluid  containing  50  per  cent,  glycerin,  and 
it  can  be  kept  in  corked  bottles.  Before  use  this  concentrated  mallein 
must  be  diluted  with  a  one-half  per  cent,  watery  solution  of  carbolic 
acid.  The  dose  of  the  dilute  mallein  for  a  horse  is  2.5  c.c.  The 
injection  is  made  either  in  front  on  the  thorax  or  on  one  side  of  the 
neck  under  aseptic  precautions  and  with  a  sterile  syringe.  It  is  best 
to  shave  closely  the  place  where  the  injection  is  made.  Before  making 
the  mallein  test  the  temperature  of  the  animal  should  be  taken  three 
times  a  day,  morning,  noon,  and  evening,  for  two  days.  Horses  with 
fever  do  not  give  accurate  results  with  the  mallein  test.  After  the 
injection  is  made  the  animal  should  be  kept  quiet  in  a  well-protected 
stable,  with  fairly  even  temperature;  it  should  not  be  overfed  nor  per- 
mitted to  drink  large  amounts  of  cold  water.  From  six  to  sixteen  hours 
after  the  injection  the  temperature  is  to  be  taken  every  hour,  and  from 
then  to  the  thirty-sixth  hour  to  the  forty-second  hour  every  two  hours, 
with  the  exception  of  a  night's  omission,  for  about  eight  hours.  These 
strict  rules  cannot  always  be  observed,  but  the  temperatures  should  be 
taken  at  least  as  follows :  Three  times  before  the  injection;  then  every 
two  hours  for  six  to  twenty  hours  after  the  injection.  It  is  well  to  give 
the  injection  very  late  at  night,  so  that  the  taking  of  the  temperature 
may  be  begun  every  two  hours  very  early  the  next  morning. 

Effect  of  the  Mallein  Test  upon  a  Horse  not  Having  Glanders. — 
The  temperature  frequently  rises  after  a  few  hours,  but  rarely  reaches 
40°  C.,  and  goes  down  to  normal  a  few  hours  afterward.  Locally 
there  may  be  very  little  reaction,  or  there  may  be  a  slight  painless 
edema,  which  disappears  within  twenty-four  hours  after  the  injection. 

Effect  of  the  Mallein  Test  upon  a  Horse  Affected  with  Glanders.— 
The  temperature  begins  to  rise  six  to  eight  hours  after  the  injection; 
the  curve  then  rises  rapidly  during  the  next  six  to  eight  hours,  and 
reaches  its  maximum  with  40°  to  42°  C.  This  maximum  is  kept 
with  some  slight  variations  for  another  eight  hours,  and  then  the 
temperature  gradually  sinks.  Some  elevation  of  temperature  generally 
remains  twenty-four  hours  after  the  injection.  On  the  second  day 
a  similar  rise  of  temperature  occurs;  as  a  rule  it  is  not  as  intense  as 
on  the  first  day,  but  occasionally  it  is  more  so.  The  rise  in  temperature 
sometimes  appears  soon  after  the  injection  (1  hour);  sometimes  it  is 
considerably  delayed  (twenty-two  hours).  The  local  reaction  at  the 
site  of  the  injection  is  very  characteristic.  It  makes  its  appearance 
in  six  to  eight  hours  and  consists  in  a  very  painful,  well  circumscribed, 
rather  firm,  doughy  hot  swelling  about  four  to  six  inches  in  diameter. 
Later  the  boundaries  of  the  swelling  become  more  diffuse  and  less 


THE  MALLEIN  TEST  313 

sharply  defined.  The  pain  generally  ceases  or  becomes  less  marked 
on  the  second  day,  but  the  infiltration  spreads,  often  to  the  extent  of 
ten  inches  or  even  more  in  diameter.  Only  after  three  to  eight  days 
does  the  last  trace  of  the  swelling  disappear.  The  other  symptoms, 
such  as  malaise,  loss  of  appetite,  weakness,  are  inconstant  and  depend 
largely  upon  the  height  of  the  fever  and  upon  individuality.  Some 
horses  infected  with  glanders  become  very  irritable  and  sick  after  the 
injection  of  a  full  dose  of  mallein.  The  dose  varies  in  the  different 
preparations,  and  is  generally  unmistakably  indicated  on  the  label. 
It  is,  of  course,  easy  to  distinguish  between  an  absolutely  negative 
and  a  typical  fully  developed  positive  reaction,  but  cases  occur  in 
practice  in  which  the  decision  is  difficult.  Horses  very  much  advanced 
in  glanders  sometimes  do  not  react  typically,  but  in  such  cases  a 
mallein  test  is  generally  unnecessary  as  the  diagnosis  can  be  made 
from  the  clinical  symptoms. 

The  Eighth  International  Veterinary  Congress,  Buda-Pesth,  1905, 
adopted  the  following  rules  for  the  mallein  test  and  its  interpretation : 

"1.  Unless  the  results  following  the  injection  of  mallein  exhibit  the 
characteristics  of  a  typical  reaction  they  must  not  be  regarded  as 
indicating  the  existence  of  glanders. 

"2.  A  typical  reaction  comprises  a  rise  in  temperature  of  at  least 
2°  C.  The  rise  should  extend  above  40°  C.  (104°  F.).  During  the 
course  of  the  first  day  the  temperature  curve  usually  exhibits  a  plateau 
of  two  peaks,  and  on  the  second,  and  even  on  the  third,  a  more  or 
less  marked  rise.  This  rise  in  temperature  is  accompanied  by  a  local 
and  general  reaction. 

"3.  Any  rise  in  temperature  which  falls  short  of  40°  C.  (104°  F.), 
and  higher  atypical  reactions,  necessitates  a  second  test. 

"4.  A  gradual  rise  of  temperature  sustained  for  some  time  is  in- 
dicative of  glanders,  even  though  it  differs  from  the  ordinary  type 
of  diagnostic  reaction. 

"5.  The  local  typical  infiltration  at  the  point  of  injection  is  a 
certain  indication  of  glanders,  even  when  rise  in  temperature  and  the 
general  organic  reaction  fail. 

"6.  Animals  which  have  undergone  the  mallein  test,  whether  or 
not  without  reaction,  should  always  be  tested  a  second  time  after  an 
interval  of  ten  to  twenty  days. 

"7.  The  preparation  of  mallein  should  only  be  intrusted  to  scien- 
tific Government  institutions,  or  to  institutions  recognized  and  con- 
trolled by  the  State. 

"8.  With  the  object  of  determining  the  full  value  of  mallein,  and 
of  clearing  the  many  still  unexplained  points  in  regard  to  the  mallein 
reaction,  the  Congress  requests  the  various  European  Governments 
to  appoint  committees  to  study  the  question." 

In  testing,  mules  double  doses  of  mallein  (2  X  2.5  c.c.  =  5  c.c.) 
should  be  used;  also  in  retesting  after  an  interval  of  a  few  weeks  in 
doubtful  cases. 


314  GLANDERS  BACILLUS 

Choromausky  has  recommended  a  so-called  ophthalmo  test  for 
glanders.  It  consists  in  the  instillation  of  a  very  small  amount  of 
mallein  into  the  conjunctival  sac  of  the  eye.  According  to  Wladimiroff 
this  reaction  is  not  very  accurate,  since  horses  free  from  glanders  may 
also  react  positively  by  an  inflammatory  hyperemia  of  the  conjunctiva. 

Agglutination  Test. — If  the  blood  serum  of  horses  suffering  from 
glanders  is  allowed  to  act  on  a  young,  uniformly  cloudy  bouillon 
culture  of  glanders  bacilli  the  latter  becomes  agglutinated,  form  small 
lumps,  and  these  fall  to  the  bottom  of  the  test-tube  or  the  watch-glass 
in  which  the  test  is  made.  (McFadyean.)  This  change  is  due  to 
the  precipitins  and  agglutinins  present  in  the  blood  serum  of  gland- 
erous horses.  Such  substances,  however,  are  also  present  in  the 
blood  of  healthy  horses,  but  in  much  smaller  amounts,  and  for  this 
reason  the  agglutination  test  in  glanders  has  to  be  made  as  a  quanti- 
tative test.  As  a  rule,  serum  from  a  healthy  horse  will  agglutinate  in 
a  strength  of  1  in  200  to  300  or  even  400;  while  blood  serum  from  a 
glandered  horse  will  agglutinate  the  bacilli  in  a  dilution  of  1  in  1000, 
1500,  and  even  more. 

From  a  large  number  of  tests  made  on  healthy  animals  and  on 
horses  suffering  from  glanders  the  following  rules  may  be  drawn: 

Horses  whose  blood  agglutinates  only  in  dilution  up  to  500  are 
probably  healthy,  yet  among  them  occur  a  few  cases  (about  6  per 
cent.)  of  glanders. 

Horses  agglutinating  in  a  dilution  up  to  800  are  probably  affected 
with  glanders,  yet  about  3  per  cent,  of  them  are  free  from  this  disease. 

Horses  whose  blood  serum  agglutinates  in  dilutions  up  to  1000  or 
more  are  surely  infected  with  glanders. 

Parke,  Davis  &  Company  have  devised  an  apparatus  called  by 
them  the  Glanders  Agglutometer,  which  very  much  simplifies  the 
agglutination  test  for  the  veterinary  practitioner.  It  includes  an 
emulsion  of  killed  glanders  bacilli  and  circular  pieces  of  filter  paper 
varying  in  size  for  procuring  a  fairly  definite  amount  of  serum. 
Various  sizes  of  filter  paper  are  dipped  into  the  serum  to  be  tested, 
and  are  then  placed  in  small  test-tubes,  which  are  filled  up  to  a  mark 
with  the  bacterial  emulsion.  In  this  manner  dilutions  of  1  in  200, 
1  in  500,  1  in  800,  and  1  in  1200  are  obtained.  The  tubes  are  set 
aside  in  a  warm  place  for  a  number  of  hours  and  then  inspected.  It  is 
noted  in  what  dilutions  agglutination  and  precipitation  has  occurred. 

Whenever  an  agglutination  test  is  made  it  is  necessary  to  draw 
some  blood  from  the  suspected  horse;  this  is  best  done  from  the 
j  ugular  vein,  with  a  sterile  syringe.  The  blood  may  then  be  allowed 
to  coagulate  spontaneously,  and  after  several  hours  the  serum  can  be 
removed,  or  the  latter  may  be  separated  and  collected  at  once  by  the 
aid  of  a  centrifuge. 

The  Value  of  Mallein  as  a  Diagnostic  and  as  a  Curative. — The  value 
of  mallein  as  a  diagnostic  of  latent  and  occult  glanders  in  equines 
has  been  established  beyond  doubt.  It  has  become  the  most  important 


PSEUDOGLANDERS  315 

agency  in  the  campaign  to  stamp  out  glanders  and  to  protect  healthy 
animals  against  the  danger  of  infection  from  sick  ones.  If  all  the 
horses  of  a  stable,  company,  or  landowner  are  at  intervals  subjected 
to  the  mallein  test,  immediately  upon  acquisition  and  a  few  times 
thereafter  at  periods  of  six  to  twelve  months,  glanders  can  be  entirely 
suppressed  and  kept  out.  Before  the  general  use  of  the  mallein  test 
this  was  impossible,  because  the  disease  often  takes  a  very  chronic 
and  latent  course,  and  a  number  of  animals  may  be  infected  from  a 
single  sick  one  before  the  disease  can  be  safely  detected  by  manifest 
clinical  symptoms.  Since  the  glanders  bacillus  is  a  strict  parasite 
and  cannot  exist  for  any  great  period  external  to  the  body  of  sus- 
ceptible beings,  the  stamping  out  of  the  disease  by  united,  systematic 
and  rational  efforts,  should  be  accomplished  within  a  short  time. 
Whether  mallein  used  in  repeated,  increasing  doses,  as  recommended 
by  Babes,  Pilavios,  MacFadyean,  has  really  a  curative  effect  in 
glanders  is  a  question  which  has  not  yet  been  settled  definitely,  though 
Nocard  and  others  have  reported  a  number  of  such  cases  in  which  a 
cure  evidently  followed  the  systematic  use  of  mallein. 


PSEUDOGLANDERS. 

Several  other  infections  in  horses  not  only  clinically,  strongly 
simulate  glanders,  but  the  causative  microorga'nisms  are  pathogenic 
to  guinea-pigs  and  act  very  much  like  the  glanders  bacillus,  so  that 
an  erroneous  diagnosis  is  easily  possible  unless  the  mallein  test  is 
employed.  One  of  these  diseases  in  horses  is  known  as  lymphangitis 
ulcer osa  (pseudofarcinosa).  This  disease,  comparatively  common  in 
France,  has  been  studied  extensively  by  Nocard,  who,  in  1892,  dis- 
covered as  its  specific  cause  a  bacillus  now  known  under  the  name 
of  Bacillus  lymphangitidis  ulcerosa.  Clinically  the  disease  is  char- 
acterized by  cutaneous  abscess  formation,  suppurative  ulcers,  and 
swelling  of  subcutaneous  lymph  glands,  a  picture  resembling  skin 
glanders  or  farcy.  The  lesions  do  not  remain  localized  in  the  skin  or 
in  the  subcutaneous  connective-tissue  lymph  glands,  but  deeper  glands 
become  involved,  particularly  those  of  the  inguinal  region,  those  along 
the  seminal  cord,  those  of  the  perineal  region,  and  finally  the  kidneys 
themselves  become  the  seat  of  abscesses.  In  fatal  cases  the  lungs 
also  show  metastatic  abscesses. 

The  bacillus  of  ulcerative  lymphangitis  in  the  horse  is  found  in 
large  numbers  in  the  pus.  It  is  rather  a  plump  and  short  rod,  with 
rounded  ends,  often  ovoid  and  broader  in  the  middle,  also  sometimes 
club-shaped.  It  is  Gram  positive.  It  can  be  cultivated  best  in  the 
incubator  at  30°  to  40°  C.,  and  does  not  grow  at  room  temperature. 
In  nutrient  bouillon  it  forms,  after  three  days,  a  whitish  granular 
sediment,  while  the  supernatant  fluid  becomes  clear.  A  delicate 
pellicle  is  sometimes  formed  on  the  surface.  The  growth  in  glycerin 


316  GLANDERS  BACILLUS 

bouillon  is  quite  abundant.  On  agar  small,  whitish,  opaque  colonies 
round  or  wavy  in  outline,  are  formed.  These,  after  a  few  days, 
become  confluent  and  cover  the  medium  with  a  delicate,  moist,  easily 
detachable  growth.  On  potatoes  a  scanty,  dirty  grayish,  dry,  dusty 
growth  occurs.  The  best  medium  is  coagulated  blood  serum  on  which 
small,  round,  distinctly  margined  colonies,  elevated  at  the  centre,  are 
formed.  These  colonies  later  form  racemose  processes.  On  horse's 
serum  the  color  is  whitish,  on  cattle  serum  more  yellowish,  sometimes 
as  intense  in  color  as  colonies  of  the  Staphylococcus  pyogenes  aureus. 
The  organism  is  strictly  aerobic.  It  does  not  change  the  reaction  of 
the  medium  and  does  not  grow  well  in  milk,  which  it  does  not  coagulate. 
Cultures  remain  virulent  for  three  or  four  months.  The  bacteria  are 
killed  in  less  than  fifteen  minutes  at  65°  C.  and  in  one  hour  at  58°  C. 
Nocard  was  able  to  produce  the  typical  disease  in  two  horses  by  the 
subcutaneous  inoculation  of  two  drops  of  a  culture.  Guinea-pigs 
inoculated  subcutaneously  develops  large  abscesses  in  four  to  five 
days;  these  heal  slowly  with  scar  formation  while  new  abscesses 
develop  in  the  neighborhood. 

A  small  amount  of  pure  culture  or  pus  from  a  horse  inoculated 
into  the  peritoneal  cavity  of  a  male  guinea-pig  produces  an  orchitis 
similar  to  that  produced  by  the  inoculation  of  glanders  bacilli. 
Between  the  third  and  the  fifth  day  the  scrotum  becomes  inflamed, 
edematous,  hot,  tender,  and  painful.  Death  occurs  after  six  to  eight 
days.  Sometimes  the  orchitis  is  relatively  moderate,  and  death  does 
not  occur.  The  testicles  may  become  entirely  destroyed  by  the 
necrotic,  suppurative  process.  Rabbits  survive  after  intraperitoneal 
injection;  subcutaneous  injections  produce  an  erysipeloid  reaction. 
These  animals  die  in  a  cachectic  condition  after  intravenous  injection. 
Mice  and  pigeons  are  susceptible,  chickens  are  not. 

Another  bacterium  found  in  the  nasal  discharges  of  horses,  which 
when  inoculated  intraperitoneally  into  male  guinea-pigs  produces 
an  orchitis  and  kills  the  animals  in  four  to  five  days,  is  a  bacillus 
discovered  by  Kutscher.  This  organism  is  also  fatal  to  mice  when 
inoculated  subcutaneously.  These  bacilli,  morphologically,  are  very 
much  like  the  Bacillus  mallei,  but  they  are  Gram  positive,  while  the 
glanders  bacillus  is  Gram  negative.  The  bacillus  of  Kutscher  also 
differs  from  the  Bacillus  mallei  in  that  it  liquefies  gelatin.  On  blood 
serum  it  forms  a  deep  orange  pigment  and  on  potatoes  a  thin,  gray, 
dry  growth. 

Pseudofarcy  due  to  a  microorganism  of  the  saccharomyces  or 
blastomyces  type  is  discussed  in  a  later  chapter. 


QUESTIONS  317 


QUESTIONS. 

1.  What  are  the  other  names  for  equine  glanders? 

2.  What  does  malleus  or  morve  mean?    What  "Rotz"? 

3.  What  animals  are  naturally  susceptible  to  the  disease?     What  form  of 
glanders  is  found  in  cattle  ? 

4.  What  microorganism  is  the  cause  of  glanders?    Who  discovered  it*? 

5.  Where  is  the  disease  found? 

6.  Give  mode  of  infection  and  spread  of  glanders  in  an  infected  animal. 

7.  What  type  of  lesions  are  producedun  the  tissues  by  the  glanders  bacillus? 
(Describe  the  first  effects  in  detail.) 

8.  What  are  the  four  anatomical  types  of  glanders  lesions  in  the  horse  ? 

9.  Describe  them  in  detail. 

10.  Describe  the  pathologic  changes  in  pulmonary  glanders  in  the  horse. 

11.  Discuss  glanders  infection  in  man.     What  other  human  diseases  may  it 
be  confounded  with? 

12.  Describe  the  morphology  and  staining  properties  of  the  Bacillus  mallei. 

13.  Describe  Loeffler's  method  of  staining  glanders  bacilli  in  smears  from  pus, 
necrotic  material,  etc. 

14.  How  is  a  pure  culture  of  the  glanders  bacillus  generally  obtained  ? 

15.  Describe  its  cultural  properties. 

16.  On  what  culture  medium  is  the  Bacillus  mallei  growth  most  character- 
istic?   What  are  the  characteristic  features? 

17.  Describe  the  preparation  of  potatoes  as  a  culture  medium  for  the  Bacillus 
mallei. 

18.  WTiat  is  the  pseudomalleus  bacillus  of  Babes? 

19.  Discuss  the  resistance  of  the  glanders  bacillus. 

20.  Why  is  the  microscopic   diagnosis  of  glanders  from  simple  cover-glass 
preparations  inaccurate  ? 

21.  Describe  Strauss'  biologic  test  for  glanders,  and  state  what  is  found  when 
the  test  is  positive. 

22.  What  is  mallein?    Describe  its  preparation. 

23.  Describe  in  detail  the  mallein  test  for  glanders. 

24.  Describe  its  result  (a)  in  a  horse  free  from  glanders;  (6)  in  an  animal  suffer- 
ing from  the  disease. 

25.  Is  there  any  danger  in  subjecting  a  horse  to  the  mallein  test? 

26.  What  is  an  ophthalmo  test?    What  is  the  value  of  this  test  for  glanders 
in  horses? 

27.  Discuss  the  principle  of  the  agglutination  test  in  glanders.      Describe  its 
steps  and  the  result  in  healthy  and  in  glanders-sick  horses. 

28.  Discuss  the  diagnostic  value  of  the  mallein  test. 

29.  What  is  the  value  of   mallein  as  a  bacterine  or  vaccine  in  the  curative 
treatment  of  glanders?    Is  glanders  always  a  progressive  and  fatal  disease? 

30.  Name  an  infection  in  horses  which  simulates  glanders  and   in  which  the 
bacillus  causing  it  gives  a  positive  reaction  in  Strauss'  biologic  test  on  male 
guinea-pigs  ? 

31.  Describe  Nocard's  Bacillus  lymphangitidis  ulcerosa  and  point  out  par- 
ticularly the  features  in  which  it  differs  from  the  Bacillus  mallei. 

32.  What  is  Kutscher's  pseudoglanders  bacillus?    How  does  it  affect  a  male 
guinea-pig  when  injected  intraperitoneally  ?    What  are  the  two  most  important 
features  which  differentiate  Kutscher's  bacillus  from  the  Bacillus  mallei? 


CHAPTER    XXVII. 

BACILLUS  OF  INFECTIOUS  ABORTION— STREPTOCOCCUS  IN  ABOR- 
TION IN  MARES— STREPTOCOCCUS  OF  VAGINITIS 
VERRUCOSA  OF  CATTLE. 

BACILLUS  OF  INFECTIOUS  ABORTION. 

Occurrence  and  Historical. — Infectious  abortion,  abortus  enzooticus 
"Seuchenhaftes  Verwerfen"  (German),  "avortement  epizootique" 
(French),  is  the  name  given  to  that  form  of  abortion  in  cows  not 
due  to  accident  or  external  influences  or  to  ergot  contaminated  fodder, 
but  to  a  specific  microorganism  known  as  the  bacillus  of  infectious 
abortion  of  Bang.  That  this  form  of  abortion  in  cows  is  infectious  in 
character  was  recognized  more  than  a  hundred  years  ago  in  England. 
Frank,  Lehnert,  and  Braeuer,  between  1876  and  1880,  proved  this 
contention  by  infecting  healthy  cows  from  the  vaginal  discharges  of 
cows  which  had  aborted.  Nocard,  in  1885,  studied  the  anatomic 
changes  of  the  disease,  while  Bang,  assisted  by  Stribolt,  discovered 
the  specific  bacillus  in  1896.  Its  etiologic  relationship  to  the  disease 
was  confirmed  by  Preisz  in  1902.  Infectious  abortion  in  cows  has 
been  observed  in  various  countries  in  Europe,  also  in  the  United 
States.  It  was  studied  by  Chester,  Law  and  Moore,  who  failed 
to  find  Bang's  bacillus,  but  instead  an  organism  which  appeared  to 
belong  to  the  colon-hog-cholera  group  of  bacteria. 

Pathologic  Lesions. — According  to  Bang's  findings,  made  on 
material  very  favorable  for  the  study  of  the  affection,  the  external 
serous  surface  of  the  uterus  is  normal,  the  cervical  canal  closed  by 
tenacious  mucus.  An  abundant,  not  fetid,  exudate,  consisting  of  a 
dirty  yellowish,  thin,  mushy  material,  containing  lumpy,  mucoid 
masses  and  presenting  in  some  portions  where  most  of  the  fluid 
had  been  absorbed  a  semisolid  character,  is  present  between  the 
uterine  mucosa  and  the  ovum.  The  exudate  is  alkaline  in  reaction. 
After  its  collection  in  a  conical  glass  it  separates  into  two  layers,  an 
upper  composed  of  a  reddish-yellow  cloudy  serum  and  a  low^er  one 
of  a  thick,  dirty,  grayish  sediment.  The  chorion  upon  section  presents 
on  its  inner  surface  a  gelatinous  substance  in  which  thin  membranes 
are  found.  This  layer  is  the  delicate  connective  tissue  between 
chorion  and  allantois  in  a  condition  of  edematous  infiltration.  The 
latter  extends  to  the  fetus  and  spreads  into  it  to  the  depth  of  about 
1.5  cm.  and  also  into  the  umbilical  cord.  The  allantois  and  amniotic 
fluids  are  normal.  The  exudate  between  the  uterine  mucosa  and  the 


CULTURAL  PROPERTIES  319 

fetal  membranes  contains  the  specific  bacillus  of  Bang  in  enormous 
numbers. 

Natural  Infection. — This  is  brought  about  by  the  bull,  who  spreads 
the  disease  from  infected  to  healthy  cows.  It  is  also  believed  that 
the  infection  may  be  spread  by  contaminated  straw,  bedding,  etc. 

Artificial  Inoculation. — Pure  cultures  of  the  bacillus  inoculated  into 
the  vagina  and  intravenously,  as  practised  by  Bang,  caused  abortion 
in  cows  and  sheep  and  in  a  mare. 

Morphology. — If  a  cover-glass  preparation  is  made  from  the  exudate 
between  the  uterine  mucosa  and  the  fetal  membranes  very  character- 
istic bacteria  are  found  in  great  numbers.  Many  of  them  are  free 
between  cells,  many  others  are  found  in  cells  in  such  masses  that  the 
cell  bodies  are  much  swollen  and  extended.  These  intracellular 
bacteria  are  sometimes  so  dense  that  the  cell  itself  has  become  un- 
recognizable; at  other  times,  however,  the  nucleus  can  still  be  demon- 
strated. The  organisms  on  first  sight  appear  like  cocci,  but  a  more 
careful  exaimnation  shows  them  to  be  small,  short,  unequally  staining 
bacilli.  Some  of  them  are  as  large  as  the  tubercle  bacillus,  others  are 
quite  short  and  coccus-like.  The  unequal  staining  depends  upon  the 
presence  of  one,  two,  or  three  deeper  staining  granules.  The  organism 
is  Gram  negative. 

Cultural  Properties. — Pure  cultures  of  the  bacillus  can  be  obtained 
on  a  special  medium  devised  by  Stribolt,  which  is  prepared  as  follows : 
The  basis  is  formed  by  the  ordinary  slightly  alkaline  nutrient  bouillon 
to  which  f  per  cent,  agar  has  been  added.  This  medium  is  then 
filtered  clear  and  5  per  cent,  gelatin  is  added.  The  reaction  is  again 
corrected,  and  after  a  clear  product  has  been  obtained  it  is  distributed 
to  test-tubes  which  are  sterilized  by  the  fractional  method  and  finally 
cooled  down  to  45°  C.,  when  about  one-half  the  quantity  of  sterile 
fluid  blood  serum  (also  warmed  to  45°  C.)  is  added.  A  tube  is  then 
inoculated  from  the  material  containing  the  organisms  and  dilutions 
to  other  tubes,  are  made  in  the  usual  manner.  Tube  No.  2  is  inoculated 
three  times  from  tube  No.  1  with  a  sterile  platinum  loop,  tube  No.  3  in 
the  same  manner  from  tube  No.  2,  etc.  The  inoculated  tubes  are  then 
rapidly  cooled  in  cold  water.  When  tubes  so  inoculated  are  kept' in  the 
incubator  at  blood  temperature  very  small  punctiform  or  sometimes 
slightly  larger  round  colonies  are  formed.  These  are  situated  about 
J  cm.  below  the  upper  surface  and  extend  from  1  to  1.5  cm.  down. 
Thus  growth  takes  place  in  a  very  definite  zone,  but  neither  above 
nor  below  it.  The  bacillus  does  not  grow  in  gelatin-agar;  it  grows 
very  scantily  in  a  5  per  cent,  glycerin  bouillon,  but  better  in  a  mixture 
of  two  parts  of  such  a  glycerin  bouillon  to  which  one  part  of  serum 
has  been  added.  In  fluid  media  a  granular  sediment  is  formed  after 
several  days.  The  relation  of  the  bacillus  to  oxygen  is  peculiar.  It 
does  not  grow  under  strictly  anaerobic  conditions.  It  grows  better 
in  glycerin  bouillon  if  pure  oxygen  is  conducted  through  the  fluid,  and 
if  afterward  the  opening  of  the  tubes  or  flasks  are  sealed  air-tight 


320  BACILLUS  OF  INFECTIOUS  ABORTION 

with  paraffin.  While  the  bacillus  in  ordinary  air  does  not  grow  up 
"to  the  surface  of  the  solid  culture  media,  the  presence  of  an  atmosphere 
rich  in  oxygen  favors  growth  and  makes  it  much  more  abundant. 
This  is  true  even  when  an  excess  of  carbon  dioxide  of  from  4  to  5  per 
cent,  is  present.  Bang  found  two  optima  of  growth,  one  in  the  presence 
of  atmospheric  air,  and  one  in  the  presence  of  almost  100  per  cent, 
oxygen.  If  the  air  above  the  solid  culture  medium  is  rarefied  to  a 
certain  degree  the  growth  reaches  to  the  surface;  if  the  rarefication 
becomes  excessive,  growth  ceases  entirely. 

MacNeal  and  Kerr  have  recently  found  the  bacillus  of  contagious 
abortion  in  some  cases  of  abortion  in  cows  in  Illinois.  In  order  to 
isolate  this  peculiar  microorganism,  they  have  made  use  of  a  new 
plate  or  Petri  dish  method  devised  by  Nowak,  which  is  as  follows: 
Ordinary  agar  is  melted  and  cooled  to  50°  C.;  then  mixed  with 
about  one-fourth  its  volume  of  naturally  sterile  blood  serum,  and 
poured  into  sterile  Petri  dishes,  where  it  is  allowed  to  solidify.  The 
piece  of  placenta  or  other  material  from  the  abortion  is  streaked  over 
a  number  of  Petri  dishes  in  the  manner  generally  employed  in  preparing 
streak  dilution  cultures.  The  plates  are  then  incubated  for  twenty- 
four  hours  at  37°  C.,  to  allow  contaminating  aerobic  bacteria  to 
develop.  Colonies  which  have  developed  after  twenty-four  hours  are 
marked  with  a  ring  on  the  glass  by  the  aid  of  a  glass  pencil,  India 
ink,  or  a  cut-out  label  etc.  The  plates  are  then  put  into  an  anatomic 
jar,  Novy  jar,  or  desiccator,  with  a  culture  of  the  Bacillus  subtilis. 
About  one  square  centimeter  of  subtilis  culture  is  to  be  used  for  each 
15  c.c.  of  air  volume  of  the  jar.  The  jar  is  then  closed  and  kept  in 
the  incubator  for  three  days.  The  growth  of  the  subtilis  bacillus  is 
used  to  absorb  some  of  the  oxygen  of  the  air  in  the  jar  and  to  bring 
about  those  conditions  which  favor  the  growth  of  the  abortion  bacillus. 
If  any  of  these  are  present  they  develop  as  transparent  colonies  with 
the  characteristics  already  described. 

Resistance. — The  bacillus  dies  out  rapidly  in  pure  cultures  (in  about 
two  weeks).  It  is  killed  at  55°  C.  in  three  minutes,  in  corrosive 
sublimate  1  to  2000  in  fifteen  seconds,  in  1  per  cent,  carbolic  acid 
in  one  minute,  in  2  per  cent,  acetic  acid  in  two  minutes,  and  in  1  per 
cent,  acetic  acid  in  twenty  minutes.  It  can  remain  alive  and  virulent 
in  dried  uterine  exudate  where  it  had  its  natural  habitat,  in  the 
uterine  cavity,  and  in  dead  embryos  for  many  months. 

Diagnosis. — McFadyean  and  Stockman  have  recently  studied  the 
distribution  and  diagnosis  of  epizootic  abortion  in  Great  Britain. 
They  ascertained  the  existence  of  the  disease  in  fifty-five  farms, 
distributed  over  thirty-six  counties.  They  examined  51  feti  and  found 
the  bacillus  discovered  by  Bang  in  22  feti.  In  35  fetal  membranes 
they  obtained  the  bacillus  in  33  specimens,  while  they  failed  in  only  2; 
hence  they  consider  the  examination  of  the  membranes  for  the 
presence  of  the  Bang  bacillus  a  more  trustworthy  procedure  than  the 
bacterial  examination  of  the  fetus.  The  two  investigators  attempted 


OSTERTAG'S  STREPTOCOCCUS  IN  ABORTION  IN  MARES     321 

to  work  out  an  accurate  method  of  diagnosis  by  means  of  vaccine  or 
serum  tests.  An  agglutination  test  was  devised,  based  upon  the  same 
principle  and  technique  as  the  agglutination  test  for  glanders.  It 
was  found,  however,  that  the  difference  between  the  agglutinating 
powers  of  the  serum  of  a  cow  affected  with  abortion  and  that  of 
normal  animals  was  too  slight  to  inspire  confidence  in  such  a  test. 
It  was  found  during  these  experiments  that  the  milk  of  an  animal 
which  had  aborted  possessed  agglutinating  properties  up  to  1  to  25, 
but  owing  to  the  opacity  caused  by  the  addition  of  milk  to  a  culture 
the  lacteal  fluid  was  found  unsuitable  for  such  agglutination  tests. 
Another  test  tried  was  one  based  upon  the  principle  of  complement 
fixation,  which  has  found  such  wide  application  in  the  diagnosis 
of  latent  human  syphilis  (see  Chapter  VII).  The  serum  for  this 
test  for  epizootic  abortion  is  obtained  from  blood  drawn  from  the 
jugular  veins  of  the  cows.  The  antigen  is  prepared  from  a  pure 
culture  of  the  abortion  bacillus  emulsified  in  physiologic  salt  solution. 
The  complement  is  derived  from  the  serum  of  guinea-pig's  blood; 
the  hemolytic  amboceptor  from  the  serum  of  goats  sensitized  for 
ox-blood  corpuscles,  and  the  latter  were,  of  course,  used  for  the  final 
hemolytic  test.  As  controls  the  blood  serum  of  healthy  cows  and  of 
cows  infected  artificially  with  the  abortion  bacillus  were  used.  Un- 
fortunately, the  results  of  these  complicated  and  painstaking  tests 
of  the  authors,  the  first  to  apply  the  principle  of  complement  fixation 
to  the  diagnosis  of  an  animal  disease,  were  not  uniform  and  trust- 
worthy. The  last  series  of  experiments  for  the  diagnosis  of  epizootic 
abortion  was  made  with  a  vaccine  prepared  according  to  the  principles 
which  underly  the  preparation  of  tuberculin  and  mallein.  This 
abortin,  derived  from  pure  cultures  of  the  abortion  bacillus,  was 
injected,  and  the  following  conclusions  as  to  its  value  as  a  diagnostic 
were  drawn:  "It  would  appear  that  a  rise  of  temperature  to  104°  F. 
or  more  after  the  injection  of  abortin  may  possibly  be  indicative  of 
infection,  but  it  will  be  necessary  to  carry  out  a  large  number  of  tests 
in  practice  before  deciding  upon  the  value  of  the  method  of  diagnosis." 


OSTERTAG'S  STREPTOCOCCUS  IN  ABORTION  IN  MARES. 

An  extensive  epizootic  of  abortion  among  mares  was  studied  in 
1899  and  1900  by  Ostertag.  While  he  expected  to  find  as  its  cause 
Bang's  bacillus,  he  failed  entirely  in  spite  of  the  use  of  the  proper 
culture  medium  to  encounter  this  organism,  but  instead  of  it,  he 
obtained  from  the  edematous  fluid  between  the  uterine  mucosa  and 
the  fetal  membranes,  from  the  heart's  blood,  the  pleural  fluid,  and  the 
gastric  contents  of  the  dead  feti  a  short,  immobile,  Gram-negative 
streptococcus.  The  organism  was  difficult  to  cultivate;  it  grew  best 
in  serum  bouillon,  serum  agar,  and  the  transudate  of  dead  feti.  The 
fluid  media  are  clouded  by  these  growing  streptococci  in  two  days, 
21 


322  BACILLUS  OF  INFECTIOUS  ABORTION 

and  after  two  more  days  a  sediment  is  formed.  On  serum  agar  the 
growth  is  very  delicate  and  scarcely  visible  to  the  naked  eye.  The 
organism  does  not  grow  on  gelatin  or  in  milk,  and  very  poorly  in 
nutrient  bouillon  without  the  addition  of  serum.  Transplants  cannot 
be  kept  up  long  on  any  medium;  they  generally  fail  after  the  fif*h 
generation.  Ostertag  injected  cultures  of  this  streptococcus  into  two 
pregnant  mares.  The  one  which  was  injected  intravenously  aborted 
after  twenty  days;  the  other  one,  inoculated  into  the  vagina,  went  to 
full  term,  but  gave  birth  to  a  very  weak  foal. 

These  streptococci  are  not  pathogenic  for  either  mice,  guinea-pigs, 
or  rabbits ;  they  are  very  slightly  resistant  to  disinfectants.  The  natural 
infection  is  brought  about  by  sexual  intercourse. 

STREPTOCOCCUS  OF  VAGINITIS  VERRUCOSA  OF  THE  COW. 

An  infectious  vaginitis  in  cows  is  relatively  prevalent  in  several 
European  countries.  It  has  been  particularly  widespread  in  Eastern 
Germany.  The  disease  is  known  under  the  names  of  kolpitis  granulosa, 
infectiosa  bovum,  vaginitis  verrucosa;  "Ansteckender  Scheiden- 
katarrh  der  Kinder"  or  "Knotchenseuche"  (German).  The  affection 
is  chronic  in  character  and  does  not  yield  easily  to  treatment.  When 
healthy  cows  are  infected  experimentally  from  the  vaginal  discharges 
of  sick  animals,  swelling,  redness,  and  tenderness  of  the  vaginal  mucosa 
appear  after  two  to  three  days.  Afterward  the  lymph  follicles  of  the 
mucous  membrane  swell  up  and  form  granules.  It  is  this  change 
which  has  led  to  the  name  granular  vaginal  catarrh  of  the  cow. 
Ostertag  and  Hecker  discovered  as  the  cause  of  the  disease  a  short 
streptococcus  of  generally  six  to  nine  individual  cocci,  which  are 
surrounded  by  a  capsule.  This  streptococcus  is  found  not  only  in 
the  discharge,  but  it  penetrates  deep  into  the  epithelial  layers  and 
into  the  papillae  of  the  vaginal  mucosa.  The  organism  stains  best 
with  Loeffler's  alkaline  methylene  blue;  it  is  Gram  negative.  The 
streptococcus  can  be  easily  cultivated  on  glycerin  agar,  coagulated 
blood  serum,  in  gelatin,  and  in  bouillon.  The  latter  becomes  diffusely 
cloudy.  The  organism  does  not  liquefy  gelatin  or  coagulated  blood 
serum;  it  does  not  coagulate  milk,  nor  form  hydrogen  sulphide,  indol, 
or  gas  in  media  containing  glucose. 

Animals  Susceptible. — The  ordinary  laboratory  animals  are  not  sus- 
ceptible to  infection  with  this  organism.  Infected  vaginal  discharges 
of  cows  or  pure  cultures  of  the  streptococcus  produce  the  typical 
disease  in  healthy  cows,  if  inoculated  into  their  vaginae.  Sheep,  goats, 
horses,  and  hogs  cannot  be  infected  in  this  manner.  In  addition 
to  the  specific  streptococcus  the  vaginal  discharges  of  cows  suffering 
from  the  diseases  generally  also  show  staphylococci  and  colon  bacilli. 
Bulls  spreading  the  disease  from  sick  to  healthy  cows  are  generally 
not  made  sick,  exceptionally,  however,  they  likewise  develop  a 
catarrhal  discharge  from  the  penis. 


QUESTIONS  323 

Resistance. — The  resistance  of  the  organism  to  disinfectants  is  of  a 
very  low  degree,  yet  its  eradication  in  an  infected  vagina  is  difficult 
because  it  penetrates  deeply  into  the  tissues  of  the  mucosa. 


QUESTIONS. 

1.  What  kind  of  disease  is  abortus  enzooticus  of  cattle? 

2.  To  what  is  it  due?    Who  discovered  its  microorganisms? 

3.  Describe  the  characteristic  pathologic  lesions  of  the  affection. 

4.  Describe  Bang's  abortion  bacillus  as  found  in  the  exudate  between  the 
uterine  mucosa  and  the  fetal  membranes. 

5.  How  is  the  disease  spread  under  natural  conditions? 

6.  What  animals  are  susceptible  to  artificial  inoculation? 

7.  Describe  the  morphology  of  the  bacillus  of  infectious  abortion. 

8.  Describe  the  preparation  of  Stribolt's  gelatin-agar-serum  mixture. 

9.  How  does  the  Bang  bacillus  grow  in  it? 

10.  What  are  the  relations  of  the  growth  of  this  organism  to  oxygen  ? 

11.  Discuss  the  resistance  of  the  bacillus. 

12.  Discuss  the  diagnosis  of  the  disease. 

13.  What  is  the  cause  of  infectious  abortion  in  mares?     Describe  the  organism. 

14.  Describe  its  cultural  properties. 

15.  What  is  the  cause  of  infectious  granular  vaginitis  in  cows? 

16.  Describe  the  pathologic  lesions  in  kolpitis  granulosa  infectiosi  bovum. 

17.  Why  is  the  disease  comparatively  resistant  to  treatment? 

18.  Describe  the  morphologic  and  cultural  properties  of  the  streptococcus  of 
bovine  vaginitis. 


CHAPTEK    XXVIII. 

TUBERCULOSIS— DISTRIBUTION   AMONG  MAN   AND   ANIMALS- 
ROUTES  OF  INFECTION. 

THE  term  tuberculosis  is  derived  from  the  Latin  word  tuberculum, 
the  diminutive  of  tuber,  which  means  a  knob,  protuberance,  or  nodule. 
It  is  a  very  widespread  disease  among  man  and  some  of  the  domestic 
animals,  particularly  cattle  and  swine,  and  is  characterized  anatom- 
ically by  the  formation  of  poorly  vascularized  or  avascular  nodules, 
which  have  a  tendency  to  become  caseous.  The  disease  in  all  of  its 
forms  is  due  to  a  specific  organism  known  as  the  tubercle  bacillus  of 
Robert  Koch. 

Historical  Remarks. — The  pulmonary  form  of  tuberculosis  of  man 
is  also  termed  consumption,  or  phthisis,  i.  e.,  a  consuming  or  wasting 
away,  and  has  been  known  to  mankind  for  thousands  of  years.  A 
vivid  description  of  the  disease  is  found  in  the  works  of  Hippocrates, 
but  it  would  hardly  have  enabled  the  Father  of  Medicine  to  pass  a 
very  creditable  examination  as  to  etiology  and  morbid  anatomy. 
Hippocrates1  distinguished  three  forms  of  pulmonary  tuberculosis 
in  man,  and  his  description  is  so  highly  interesting  that  a  few  passages 
from  it  will  not  be  out  of  place.  He  says:  "There  are  three  kinds 
of  pulmonary  consumption.  The  first  kind  is  due  to  mucus.  If  the 
head,  filled  with  mucus,  becomes  diseased  and  heated  the  mucus  in 
the  head  becomes  putrid  because  it  cannot  be  removed  properly. 
After  the  mucus  has  become  condensed  and  putrid,  and  after  the 
bloodvessels  have  become  overfilled  with  it,  a  flow  toward  the  lungs 
is  established,  and  as  soon  as  the  lungs  contain  this  mucus  they  become 
corroded  because  the  latter  is  salty  and  turbid.  The  patient  now 
experiences  a  mild  fever,  with  chills,  the  chest  and  the  back  are 
painful,  he  is  tortured  by  a  violent  cough,  and  he  coughs  up  large 
masses  of  a  moist,  salty  sputum.  This  is  what  he  experienced  in 
the  beginning  of  the  disease,  and  in  its  farther  course  the  body  wastes, 
with  the  exception  of  the  legs,  which  swell  up,  also  the  feet  swell,  and 
"""" Hthe  nails  become  contracted.  He  becomes  lean  around  the  shoulders, 
the  larynx  becomes  filled  with  a  kind  of  foam,  and  the  breath  whistles 
like  the  air  in  a  reed.  During  the  disease  the  patient  has  great  thirst; 
he  becomes  very  weak.f  If  he  is  in  this  conditioners  a  rule,\he  perishes 
Q  miserably  from  the  wasting  away  within  a  year. 

1  The  quotations  are  taken  from  the  German  translation  of  Hippocrates'  works,  by  Robert 
Fuchs,  Munich,  1897,  vol.  ii,  chap,  x,  p.  494  et  seq. 


HISTORICAL  REMARKS  325 

"The  second  kind  of  pulmonary  consumption  is  due  to  overwork, 
and  the  third  kind  to  an  overfilling  of  the  bone  marrow  and  blood- 
vessels, with  watery  mucus  and  bile." 

The  treatment  recommended  by  Hippocrates  is  chiefly  dietetic  and 
hygienic — asses',  cows',  and  goats'  milk,  boiled  or  raw,  honey,  etc. 
He  also  recommends  a  good  deal  of  walking  in  the  open  air,  with  care 
not  to  take  cold.  In  spite  of  treatment,  Hippocrates  says  the  disease 
generally  takes  a  fatal  course. 

It  appears  that  the  ancient  Hebrews  recognized  tubercular  lesions 
in  cattle,  because  the  Talmud  refers  to  morbid  changes  found  in 
slaughtered  cattle,  and  it  interdicted  the  use  of  meat  from  animals 
with  such  ulcerative,  evidently  tubercular,  lesions. 

After  the  writings  of  Hippocrates  little  progress  was  made  in  the 
study  of  consumption  for  many  hundreds  of  years,  but  from  time  to 
time  the  belief  that  it  was  an  infectious  disease  sprang  up,  only  to  be 
forgotten  again. 

Silvius  and  Morgagni  were  the  first  to  point  out  the  inter-relation 
between  the  formation  of  nodules  and  their  subsequent  breaking 
down  and  ulceration  in  the  production  of  pulmonary  phthisis.  Silvius 
considered  the  disease  contagious.  Bayle  and  Laennec  were  the 
first  investigators  of  tuberculosis  who  recognized  the  importance  of 
caseous  material  in  the  natural  history  of  the  disease.  Laennec  recog- 
nized the  identity  of  tuberculosis  and  what  was  called  scrofulosis. 
Virchow  established  the  anatomical  diagnosis  upon  the  basis  of  the 
poorly  vascularized  inflammatory  nodule,  which  later  on  undergoes 
caseation.  Virchow,  however,  did  not  believe  tuberculosis  in  man 
and  pearl  diseases  in  cattle  to  be  one  and  the  same  disease;  on  the 
contrary,  he  considered  pearl  disease  in  cattle  a  form  of  lympho- 
sarcoma  (i.  e.,  a  form  of  malignant  tumor  formation).  It  is  interesting 
to  note  here  that  tuberculosis  in  cattle  at  the  beginning  of  the  eighteenth 
century  was  looked  upon  as  a  form  of  syphilis,  due  to  sodomitic 
contact  with  infected  persons.  Before  this  time,  however,  and  later, 
in  spite  of  Virchow's  great  authority,  certain  investigators  held  that 
pulmonary  tuberculosis  in  man  and  cattle  were  one  and  the  same 
disease  (Gurth,  Hering,  Fuchs),  and  others  even  believed  that  pearl 
disease  of  the  peritoneum  in  cattle  and  human  tubercular  peritonitis 
were  identical. 

The  first  inoculation  experiments  with  material  believed  to  be 
tubercular  were  made  by  Kerwin  (1789),  Lepelletier,  Goodlad,  and 
Desgallieras.  The  experiments  were  undertaken  on  human  beings, 
but  they  were,  fortunately  for  them,  but  unfortunately  for  science, 
not  successful.  Klenke  (1843)  was  the  first  to  report  a  successful 
production  of  tuberculosis  by  intravenous  inoculation  of  a  rabbit 
with  tubercular  material,  but  his  work  did  not  attract  much  attention, 
and  was  pronounced  inaccurate  by  some  who  inoculated  apparently 
indifferent  material  and  produced  tuberculosis.  Villemin  presented 
his  celebrated  contribution  on  the  "  Cause  and  Nature  of  Tuberculosis 


326  TUBERCULOSIS 

and  the  Inoculation  of  the  same  from  Man  to  Rabbits,"  in  December, 
1865.  From  his  successful  experiments  he  drew  the  conclusions 
that  tuberculosis  is  a  specific  disease,  that  it  has  its  origin  in  an 
inoculable  virus,  which  can  be  transferred  successfully  from  man  to 
the  rabbit,  and  that  it  is.  a  virulent  disease,  which  should  be  classified 
with  smallpox,  scarlet  fever,  syphilis,  and  which  can  be  likened  to 
glanders.  Villemin's  inoculation  experiments  with  tubercular  material 
were  successfully  repeated  by  some;  others  apparently  produced 
tubercular  lesions  with  morbid,  but  not  tubercular,  material  and  with 
pieces  of  glass,  rubber,  wood,  etc.,  so  that  Villemin's  views  were  soon 
discredited.  The  work  of  Schueller,  Tappeiner,  Langhans,  Cohnheim, 
Salmonsen,  Baumgarten,  Klebs,  Chaveau,  Bellinger,  Kitt,  Gerlach, 
and  others,  undertaken  after  the  publication  of  Villeman's  investi- 
gations and  continued  until  the  year  1881,  gradually  demonstrated 
more  and  more  clearly  the  infectious  nature  of  tuberculosis.  Klebs, 
Toussaint,  and  others  made  attempts  to  cultivate  the  unknown  living 
virus  of  tuberculosis,  and  Aufrecht  and  Baumgarten  undoubtedly 
saw  tubercle  bacilli  which  they  were  unable  either  to  stain  or  to 
cultivate.  It  was  Robert  Koch,  however,  who  finally  succeeded  in 
establishing  the  etiology  of  tuberculosis,  this  most  important  disease 
of  man  and  many  of  the  domestic  animals. 

The  cultural  methods  devised  by  himself,  the  experiments  he  under- 
took, his  deductions  and  conclusions  drawn  from  them,  and  his  first 
formal  communication  are  even  today  a  monument  and  model  of 
classical  experimental  research  work  in  medicine.  Some  of  those 
who  have  followed  in  his  footsteps  and  profited  by  his  pioneer  work 
appear  to  have  forgotten  the  debt  owed  him  and  have  venomously 
attacked  him  for  the  view  he  took  as  to  the  intertransmissibility  of 
bovine  and  human  tuberculosis.  This  question  is  as  yet  by  no  means 
fully  settled,  but  in  the  light  of  researches  made  during  the  last  decade 
it  certainly  appears  that  Koch's  standpoint  was  too  radical. 

Distribution  in  Man. — Tuberculosis  in  human  beings  occurs  almost 
over  the  entire  world.  It  is  absent  only  at  very  high  altitudes. 
It  occurs  at  all  ages,  but  the  greatest  number  of  advanced  cases 
are  found  in  the  middle  years  of  life.  The  United  States  Census 
of  1890  showed  that  with  a  population  of  76,000,000  there  died 
in  that  one  year  111,059  persons  from  tuberculosis  of  the  lungs, 
or  about  one-ninth  of  the  deaths  from  all  causes  were  due  to  pul- 
monary tuberculosis.  In  Germany  the  mortality  was  still  larger, 
reaching  118,706  in  a  population  of  56,000,000.  These  figures 
include  only  deaths  from  the  pulmonary  form  of  tuberculosis,  and 
convey  an  incorrect  idea  of  the  prevalence  of  this  disease  among  the 
human  race,  because  pulmonary  tuberculosis  is  often  very  chronic, 
and  exists  for  a  long  time  before  it  leads  to  death,  and  tubercular 
infections  are  by  no  means  always  fatal,  as  is  frequently  believed 
among  the  laity.  On  the  contrary,  they  often  heal  spontaneously, 
and  death  results  from  different  causes  in  individuals  with  healed 


TUBERCULOSIS  IN  ANIMALS  327 

tuberculosis  or  affected  with  its  latent  form  which  may  lead  to  recovery, 
or  to  a  more  actue,  eventually  fatal  outbreak.  The  most  careful 
postmortem  examinations  on  human  material  show  that  probably 
not  less  than  70  to  90  per  cent,  of  all  persons,  no  matter  from  what 
disease  they  may  die,  have  at  some  time  had  a  tubercular  infection. 
Pulmonary  tuberculosis  has  often  been  found  absent  among  uncivil- 
ized tribes  living  in  a  state  of  nature,  but  contact  with  civilized  races 
soon  causes  it  to  appear  among  them  and  often  to  become  frightfully 
prevalent,  as  among  the  Indians  and  negroes  in  the  United  States. 
Another  good  example  of  the  same  tendency  is  found  among  the 
inhabitants  of  the  Philippine  Islands,  who  before  the  advent  of  the 
Spaniards  were  apparently  free  from  tuberculosis.  Now,  tuberculosis 
is  frightfully  prevalent  among  those  tribes  which  have  come  into 
contact  with  the  civilized  races  for  several  hundred  years.  One- 
encouraging  feature  in  regard  to  the  prevalence  of  tuberculosis  lies 
in  the  fact  that  the  mortality  and  evidently  also  the  number  of  persons 
affected  has  diminished  since  its  cause  and  manner  of  spreading  has 
become  known  and  made  precautions  and  prophylaxis  possible.  In- 
Germany  the  death  rate  per  year  per  1000  inhabitants  has  fallen 
from  3.1  to  1.9  from  1890  to  1903;  and  in  the  United  States  from 
1890  to  1900,  from  2.544  to  1.095  per  year  per  1000  inhabitants. 

Tuberculosis  in  Animals. — Tuberculosis  among  animals,  in  the  wild 
state,  both  mammalians  and  birds,  is  practically  unknown;  but  it  is 
quite  prevalent  among  domestic  animals  and  among  wild  animals 
confined  in  zoological  gardens,  menageries,  etc.  Monkeys,  which 
in  nature  are  never  found  to  be  infected  with  tuberculosis,  generally 
die  from  it  in  captivity;  guinea-pigs  and  rabbits,  which  are  the  most 
susceptible  common  laboratory  animals,  and  very  frequently  used 
in  inoculation  experiments,  are  rarely  affected  spontaneously  even 
in  captivity.  Antelopes,  giraffes,  zebras,  wild  animals  of  the  canine 
and  feline  tribes,  often  contract  the  disease  in  confinement.  Wild 
birds  in  aviaries  often  contract  the  avian  type  of  the  disease,  but 
with  the  exception  of  parrots,  rarely  the  mammalian  type. 

Tuberculosis  has  also  been  observed  in  goats,  sheep,  dogs  and  cats 
and  quite  frequently  among  domestic  fowl. 

Cattle. — Tuberculosis  among  cattle  running  wild  on  extensive  steppes 
and  prairies  is  rare,  but  when  the  animals  are  kept  in  barns,  crowded 
and  subjected  to  stable  feeding,  it  becomes  very  common.  It  reaches 
its  highest  percentage  among  milch  cows,  which  are  often  kept  under 
the  most  unnatural  and  unhygienic  conditions.  There  are  cases 
on  record  where  more  than  one-half  and  up  to  80  per  cent,  of  a  herd 
of  milch  cows  have  been  found  infected. 

Hogs. — The  disease  has  become  widespread  among  hogs  since  it 
has  become  customary  to  feed  them  on  skimmed  milk  which  has 
been  returned  from  the  creamery.  The  milk  from  a  few  cows  affected 
with  obvious  or  latent  tuberculosis,  when  mixed  with  a  large  amount 
of  milk  from  unobjectionable  sources,  may  infect  the  entire  quantity, 


328  TUBERCULOSIS 

and  by  its  return  in  small  lots  to  a  number  of  farms  may  spread 
tuberculosis  among  hogs  to  which  the  skimmed  milk  is  fed  raw. 
Mohler  and  Washburn  have  recently  dealt  with  this  subject  in  a 
paper  presented  at  the  44th  annual  meeting  of  the  American  Veterinary 
Association  in  Kansas-  City.  According  to  their  figures,  in  three 
cities  in  one  of  the  leading  dairy  States,  from  3.1  to  6.4  per  cent, 
of  all  hogs  slaughtered  were  found  affected  with  tuberculosis.  Hogs 
also  frequently  contract  tuberculosis  by  being  fed  on  the  same  premises 
with,  tuberculous  cattle,  or  by  being  allowed  to  feed  on  offal  from 
slaughter  houses  or  to  devour  the  carcasses  of  cattle  dead  from 
tuberculosis.  In  a  case  cited  by  Mohler  and  Washburn  of  a  hog 
raiser  who  fed  40  hogs  on  the  carcass  of  a  cow  dead  from  tuberculosis, 
31  of  the  40  had  to  be  condemned  for  tuberculosis  when  they  came 
to  be  slaughtered.  Hogs  also  easily  acquire  tuberculosis  when  they 
are  taken  care  of  by  tubercular  persons  who  cough  and  spread  their 
sputum  promiscuously  in  the  pigpens. 

Modes  of  Infection  and  Transmission. — It  was  formerly  believed  that 
tuberculosis  was  most  frequently  inherited  from  parents  by  the 
offspring.  This  view  has  now  been  entirely  abandoned  and  the 
inhalation  and  the  ingestion  of  tubercle  bacilli  are  recognized  as 
the  most  frequent  modes  of  infection. 

— *  Inhalation. — In  man,  tuberculosis  is  most  frequently,  and  in  adults 
almost  exclusively,  contracted  by  the  inhalation  of  tubercular  material 
from  previously  infected  individuals.  The  sputum  coughed  up  at 
frequent  intervals  by  patients  suffering  from  pulmonary  tuberculosis 
contains  many  millions  of  tubercle  bacilli.  These  may  be  inhaled 
directly  with  fine  moist  particles  floating  in  the  air  for  some  time  or 
the  sputum  may  be  dried  out  and  pulverized  and  the  dust  particles 
inhaled.  ('It  was  first  shown  by  Cornet  that  the  dust  accumulating  in 
rooms,  wards,  barracks,  prisons,  etc.,  where  consumptives  had  been 
allowed  to  spit  on  the  floor,  contained  live,  virulent  tubercle  bacilli. 
Ever  since  that  timejefforts  have  been  made  throughout  the  civilized 
world  to  prevent  consumptives  from  spreading  their  tubercular 
sputum  promiscuously  and  to  enforce  the  immediate  destruction  of 
the  infected  sputum  by  collecting  it  in  proper  receptacles  containing 
a  proper  antiseptic.  This  simple  measure  has  probably  contributed 
more  to  the  reduction  of  pulmonary  tuberculosis  than  any  other 
single  measure.  /There  are  two  theories  as  to  the  development  of 
tuberculosis  from  inhaled  tubercle  bacilli.  According  to  the  older, 
more  generally  accepted  view,  the  tubercular  process  develops  directly 
in  the  pulmonary  alveoli.  According  to  a  more  recent  view,  the 
tubercle  bacilli  are  first  absorbed  through  the  bronchial  mucosa,  taken 
up  by  the  lymphatics,  and  carried  to  the  bronchial  lymph  glands, 
where  they  multiply  and  from  which  point  they  later  invade  the 
C  parenchyma  of  the)  lungs. 

Inhalation  tuberculosis  is  also  very  common,  among  cattle.  In 
dairies  a  single  cow  coughing  up  tubercular  material  often  spreads 


MODES  OF  INFECTION  AND  TRANSMISSION  329 

it  to  other  cows.  Cows  infected  with  pulmonary  tuberculosis  also 
swallow  a  considerable  quantity  of  their  sputum  and  disseminate  the 
bacilli  through  their  feces,  which  may  contaminate  the  fodder  of 
other  cows. 

Ingestion. — This  is  the  most  common  mode  of  infection  in  hogs. 
Its  process  through  skimmed  milk  from  tubercular  cows,  feeding 
after  infected  cattle,  and  devouring  tubercular  offal  or  cadavers  has 
already  been  referred  to.  Tuberculosis  is  also  contracted  through 
the  intestinal  tract  by  calves  feeding  on  cows  with  either  more  or  less 
generalized  tuberculosis  or  with  udder  tuberculosis. 

Von  Behring  has  advanced  a  theory  that  tuberculosis  in  man  is 
almost  always  primarily  an  intestinal  infection  in  which  the  tubercle 
bacilli  are  taken  into  the  body  in  early  childhood  with  milk  from 
tubercular  cows.  The  bacilli  then  remain  latent  in  the  abdominal 
cavity,  particularly  in  the  mesenteric  glands.  Later  they  enter  the 
lymphatics  or  the  blood  circulation  and  find  the  lungs  the  most 
favorable  place  for  their  development  and  the  establishment  of  an 
extensive  tubercular  process.  Certain  statistics  have  been  produced 
in  opposition  to  von  Behring's  view.  They  show  that  among  a  large 
number  of  pulmonary  consumptives,  only  a  very  small  percentage 
was  fed  on  cow's  milk  as  infants,  while  the  great  majority  was  breast- 
fed and  had  never  even  received  very  much  cow's  milk.  A  powerful 
argument  indicating  that  pulmonary  tuberculosis  is  developed 
independent  of  feeding  with  cow's  milk  is  the  following,  which  the 
author  has  not  found  mentioned  elsewhere.  Most  monkeys  and  apes 
kept  in  zoological  gardens  die  from  pulmonary  tuberculosis.  Most 
of  these  animals  have  been  born  in  tropical  or  subtropical  countries. 
They  have,  of  course,  been  breast-fed  by  mothers  never  sick  of 
tuberculosis  in  the  wild  state,  and  have  probably,  throughout  their 
lives,  never  had  a  drop  of  cow's  milk.  Yet  in  captivity  these  animals 
almost  invariably  die  of  pulmonary  tuberculosis  which  they  contract 
by  inhaling  moist  floating  particles  of  pulverized  dust  of  dried  human 
tubercular  sputum.  Another  very  strong  argument  against  the 
ingestion  theory  of  von  Behring  is  furnished  by  the  observation  that 
certain  nations,  among  which  pulmonary  tuberculosis  is  very  prevalent, 
do  not  consume  cow's  milk  nor  keep  cattle,  tubercular  or  otherwise, 
as  domestic  animals.  The  inhabitants  of  Japan  and  the  Philippine 
Islands  which,  for  example,  are  such  countries,  keep  water  buffaloes, 
or  carabaos,  as  beasts  of  burden,  but  not  as  meat-  or  milk-producing 
animals.  There  is  no  tuberculosis  among  these  carabaos,  and  as  the 
cattle  of  the  Western  nations  are  not  kept  among  the  Eastern  nations 
named,  they  cannot  have  played  any  role  in  the  great  prevalence  of 
tuberculosis  among  the  Japanese  and  the  Filipinos,  nor  can  the  disease 
of  the  lungs  be  traced  back  to  an  early  infection  with  bovine  tubercle 
bacilli. 

Wounds. — Wounds  of  the  external  surface  play  a  relatively  un- 
important part  in  producing  tubercular  infections  in  man  and  animals. 


330  TUBERCULOSIS 

Sexual  Intercourse. — Sexual  intercourse  in  cattle,  one  of  the  par- 
ticipants having  been  affected  with  tuberculosis  of  the  sexual  organs, 
has  led  to  an  infection  of  the  healthy  animal,  but  such  observations 
have  been  very  rare.  There  is  not  the  slightest  proof  that  tuberculosis 
is  ever  spread  through  spermatozoa  from  the  male  to  the  developing 
ovum.  Tuberculosis,  however,  is  occasionally  spread  from  a  tuber- 
culous mother  to  the  offspring  through  the  placenta.  These  cases  are 
rare  in  human  beings,  but  more  common  in  cattle. 

The  question  of  intertransmissibility  of  human  and  bovine  and 
mammalian  and  avain  tuberculosis  will  be  discussed  in  another 
chapter. 

QUESTIONS. 

1.  What  is  the  derivation  of  the  word  tuberculosis?    What  is  the  cause  of 
the  disease  in  its  various  forms? 

2.  Who  furnished  the  earliest  description  of  the  symptomatology,  etc.,  of 
pulmonary  tuberculosis  ? 

3.  What  is  the  derivation  of  the  term  phthisis? 

4.  Who  made  the  first  inoculation  experiments  with  what  was  considered 
tubercular  material  on  man?    What  was  the  outcome  of  these  experiments? 

5.  Describe  the  experiments  of  Villemin  and  their  results. 

6.  Discuss  the  prevalence  of  tuberculosis  among  human  beings. 

7.  To  what  percentage  are  tubercular  lesions  found  in  man  according  to 
postmortem  statistics? 

8.  Discuss  the  prevalence  of  tuberculosis  among  uncivilized  nations  living 
in  a  wild  state.    What  if  such  nations  come  into  contact  with  civilized  people 
among  whom  tuberculosis  is  of  common  occurrence? 

9.  Is  tuberculosis  among  human  beings  on  the  increase  or  decrease? 

10.  What  is  probably  the  most  important  single  method  of  combating  the 
spread  of  tuberculosis  among  human  beings? 

11.  What  animals  suffer  from  tuberculosis? 

12.  How  is  tuberculosis  most  commonly  spread  among  human  beings? 

13.  How  most  commonly  among  cattle? 

14.  How  most  commonly  among  hogs? 

15.  Discuss  the  various  routes  of  infection  by  which  tuberculosis  invades  the 
body  of  susceptible  beings. 

16.  What  are  the  two  different  views  as  to  the  most  common  mode  of  develop- 
ment of  pulmonary  tuberculosis? 

17.  Why  is  it  improbable  that  pulmonary  tuberculosis  in  man  is  generally 
due  to  the  ingestion  in  infancy  and  childhood  of  bovine  tubercle  bacilli? 


PLATE   VIII 


Section  from  the  Tubercular  Penis  of  a  Bull. 

Indicates  position  of  a  giant  cell.     Zeiss  objective  3  mm.;  compens.  ocular  No.  6. 


CHAPTER    XXIX. 

TUBERCULOSIS  (CONTINUED)— HISTOPATHOLOGY  AND  MORBID 
ANATOMY  IN  MAN  AND  ANIMALS. 

The  Tubercles. — The  characteristic  anatomical  lesion  of  tubercu- 
losis is  the  small,  avascular,  nodular  mass  of  granulation  tissue,  called 
the  tubercle,  which  has  a  very  pronounced  tendency  soon  to  undergo 
retrograde,  degenerative  changes.  The  formation  of  the  tubercle 
can  best  be  studied  experimentally,  as  has  been  done  by  a  number 
of  investigators,  including  Cohnheim,  Baumgarten,  and  others.  It 
is  accomplished  by  injecting  finely  divided  tubercular  material  or 
tubercle  bacilli  from  a  pure  culture  which  has  been  rubbed  up  and 
diluted  with  physiologic  salt  solution  into  the  anterior  chamber  of 
the  eye  of  a  rabbit.  Small,  grayish-white,  first  perfectly  translucent 
nodules  develop  in  the  eye,  within  twelve  to  fifteen  days.  Their  size 
when  first  seen  is  perhaps  not  larger  than  a  pinhead,  becoming  larger 
during  the  next  few  days  and  at  the  same  time  less  translucent;  in 
fact,  their  centres  become  opaque.  Neighboring  nodules  become 
confluent  and  in  this  manner  larger  grayish-white  or  yellowish-opaque 
nodules  are  formed.  When  infected  tissues  are  obtained  from  a 
series  of  animals  from  day  to  day  and  properly  fixed,  embedded, 
sectioned,  and  stained,  for  microscopic  study,  they  show  first  a 
slow  multiplication  of  the  injected  tubercle  bacilli.  These,  or  rather 
their  metabolic  products  or  endotoxins,  cause  some  of  the  ordinary 
fixed  connective-tissue  cells  and  some  lymphatic  endothelial  cells  to 
proliferate  much  more  rapidly  than  they  do  under  normal  conditions. 
This  increased  rate  of  cell  multiplication  or  proliferation  is  indicated 
by  the  presence  of  a  considerable  number  of  karyokinetic  figures. 

The  new  cells  formed  under  the  stimulus  of  the  infection  with 
tubercle  bacilli  become  larger  than  the  ordinary  connective-tissue 
cells  in  normal  adult  connective  tissue.  They  possess  a  vesicular 
nucleus  and  a  rather  large,  round,  or  polygonal  body  of  protoplasm. 
They  are,  in  other  words,  the  cells  generally  known  in  histopathology 
as  epithelioid  cells.  A  number  of  investigators  claim  that  the  first 
effect  of  the  presence  and  multiplication  of  tubercle  bacilli  in  connec- 
tive tissue  is  the  cell  proliferation  just  described.  Others  hold  that  the 
very  first  effect  is  a  coagulation  necrosis  of  the  connective-tissue  cell 
in  the  closest  proximity  to  the  multiplying  tubercle  bacilli,  that  this 
necrosis  leads  to  a  moderate  transudate  with  the  migration  of  leuko- 
cytes, and  that  the  proliferation  of  the  fixed  connective-tissue  cells 
begins  only  after  this  has  occurred.  It  appears  that  the  preponderance 


332 


TUBERCULOSIS 


of  evidence  is  in  favor  of  the  former  view  that  the  stimulus  to  pro- 
liferation and  the  latter  itself  precede  the  coagulation  necrosis.  In  any 
event,  if  necrosis  occurs  first,  it  is  very  insignificant,  and  the  pro- 
liferation with  the  appearance  of  karyokinetic  figures  is  very  obvious 
and  impressive  in  the  first  stage  of  the  formation  of  the  tubercle.  In 
this  first  stage  the  tubercle  bacilli  are  found  partly  between  the  cells 
and  partly  inside  of  the  protoplasm  of  epithelioid  cells.  Whenever 
bacilli  have  gained  entrance  into  the  cell  protoplasm,  they  evidently 
cause  a  profound  disturbance  of  the  cell  metabolism.  The  bacilli- 
infected  cell  grows  and  becomes  larger  by  the  absorption  of  nutritive 
material  and  its  nucleus  divides,  but  not  the  cell  protoplasm  itself. 


Fio.  148 


Section  through  tubercular  tissue  of  pleura  of  a  horse.    (Author's  preparation.) 


This  process  continues,  and  very  large  cells  with  many  nuclei  are 
formed.  The  latter  are  frequently  distributed  around  the  periphery. 
The  bacilli  infecting  such  a  large  multinudear  giant  cell  are  generally 
found  in  the  protoplasm  at  some  distance  from  the  nuclei.  The 
protoplasm  of  the  giant  cell  early  shows  evidences  of  necrobiosis  or 
coagulation  necrosis  or  granular  degeneration.  While  such  giant 
cells  are  forming  the  epithelioid  cells  increase  in  number.  Among 
them,  and  particularly  around  them  at  some  distance  from  the  point 
where  the  bacilli  are  most  numerous,  small  round  cells  with  round, 
granular,  deeply  staining  nuclei  and  small  protoplasmic  bodies  appear. 
These  cells  are  proliferated  young  connective-tissue  cells,  which  have, 


CASSATION  333 

however,  not  attained  the  larger  size  and  the  other  characteristics 
of  the  epithelioid  cells.  These  are  called  lymphoid  cells.  While 
all  these  newly  formed  cells  appear  and  accumulate,  they  push  aside 
to  a  great  extent  the  fibrous  matrix  of  the  connective  tissue  in  which 
they  have  developed.  In  consequence  of  this  the  young  cellular 
tubercle  shows  a  rather  scanty  fibrous  reticulum.  If,  however,  the 
cell  proliferation  has  remained  within  moderate  limits,  when  few 
tubercle  bacilli  have  been  present  and  when  they  are,  perhaps,  of  a 
low  type  of  virulency,  or  when  the  process  has  come  to  a  standstill,  or 
when,  in  consequence  of  natural  or  artificial  protective  influences,  a 
process  of  healing  is  setting  in,  and  when  some  of  the  newly  formed 
inflammatory  cells  have  disappeared  in  consequence  of  softening, 
liquefaction  and  absorption,  the  fibrous  reticulum  may  become  quite 
abundant,  particularly  at  the  periphery.  During  the  formation  of 
the  tubercular  inflammatory  granulation  tissue,  in  other  words,  the 
tubercle,  there  is  a  complete  lack  of  the  formation  of  bloodvessels  in 
the  newly  formed  tissue.  In  this  respect  the  tubercle  differs  greatly 
from  ordinary  inflammatory  granulation  tissue  in  which  vascular  buds 
and  new  vessels  are  formed.  The  more  virulent  the  infecting  bacilli 
the  more  rapid  their  multiplication,  the  sooner  the  retrograde  degen- 
erative processes  become  manifest  and  the  more  extensive  they  are 
likely  to  be.  The  early  degeneration  of  the  tubercle  is  probably  not 
only  due  directly  to  the  metabolic  products  of  the  tubercle  bacillus, 
but  likewise  to  the  fact  that  new  vessels  are  not  formed  and  that 
preexisting  ones  are  obliterated  in  the  mass  of  the  newly  formed  cells 
of  the  tubercle.  For  this  reason  the  tubercle  from  the  beginning 
suffers  from  a  lack  of  proper  nutrition. 

From  the  preceeding  description  it  follows  that  a  typical  young 
tubercle,  presented  to  the  unaided  eye  as  a  small,  grayish-white, 
perfectly  translucent  nodule  generally  contains  three  kinds  of  cells, 
namely,  multinuclear  giant  cells,  epithelioid  cells,  and  lymphoid  cells, 
all  of  which  are  derived  and  descended  from  fixed  connective-tissue 
cells.  Such  young  tubercles  occasionally  also  show  some  polynuclear 
leukocytes  which  have  wandered  from  the  bloodvessels  into  the  newly 
formed  inflammatory  granulation  tissue.  Sometimes  when  the 
tubercular  infection  is  very  mild,  moderate,  and  slow  in  its  action 
upon  the  infected  connective  tissue  there  occurs  simply  an  accu- 
mulation of  and  an  infiltration  with  small  lymphoid  cells  between 
which  an  epithelioid  cell  may  be  seen  here  and  there,  particularly 
in  thin  sections.  Such  tubercles,  composed  almost  exclusively  of 
lymphoid  cells,  with  only  a  very  few  epithelioid  cells,  but  without  any 
giant  cells  whatsoever,  are  called  lymphoid  tubercles.  Unless  there  is 
other  evidence  at  hand  they  cannot  be  distinguished  from  an  ordinary 
subacute  inflammatory  small  round-cell  infiltration  or  focus. 

Caseation. — The  degeneration  of  the  tubercle  is  known  as  its  casea- 
tion,  because  there  is  formed  a  rather  dry,  but  soft,  friable  material 
of  the  consistency  and  appearance  and  other  physical  properties 


334  TUBERCULOSIS 

of  soft  cheese.  Caseation  was  looked  upon  by  Weigert  and  others  as 
a  particular  form  of  coagulation  necrosis.  It  begins,  in  fact,  in  the 
tubercle  with  the  appearance  of  the  giant  cells.  Their  protoplasm 
early  shows  evidences  of  a  coagulation  necrosis,  and  not  infrequently 
in  giant  cells  nuclear  fragments  are  found  in  a  necrotic  coarsely 
granular  protoplasm.  When  the  process  of  caseation  progresses  in 
the  giant  and  other  cells  of  the  tubercle  its  centre  becomes  completely 
necrotic.  The  cells  and  their  nuclei  break  up  into  a  granular  detritus 
which  take  both  the  eosin  stain  and  to  some  extent,  but  diffusely  or 
irregularly,  the  nuclear  stain.  During  the  formation  of  the  caseous 
material  in  the  centre  of  the  tubercle  there  is  a  certain  amount  of 
transudation  from  the  neighborhood  of  the  tubercle  and  the  necrotic 
material  becomes  infiltrated  with  a  coagulable  substance  which,  while 
not  the  true  fibrin,  is  evidently  much  like  it,  and  hence  is  called  a 
fibrinoid  substance.  The  latter  is  to  a  large  extent  responsible  for  the 
particular  rather  dry  character  and  consistency  of  the  caseous  material. 
Its  characteristics  are  also  partly  due  to  the  lack  of  vessels  and 
depending  upon  it  the  lack  of  fluids  in  the  tubercle.  The  tubercle 
which  has  undergone  caseation  in  the  centre  now  appears  somewhat 
yellowish;  it  has  lost  its  transparency  and  has  become  opaque  and 
cloudy.  At  this  stage  of  retrogressive  changes,  giant,  epithelioid,  and 
lymphoid  cells  are  seen  peripherally  to  the  caseous  centre  and  between 
them  appear  polynuclear  leukocytes  which  even  wander  into  the 
caseous  centre  itself,  where  some  of  them  may  likewise  become 
necrotic  or  may  remain  unchanged  and  active  to  perform  their 
phagocytic  function.  Still  later  the  caseous  material  becomes  softened 
and  more  creamy.  This  softening  is  probably  brought  about  by 
digestive  proteolytic  ferments,  which  are  transported  to  the  caseous 
centre  by  the  transudate  and  by  the  wandering  leukocytes.  If  by 
this  time  the  activity  of  the  bacilli  has  become  lessened,  the  develop- 
ment of  fibrous  connective  tissue  at  the  periphery  becomes  more 
abundant  and  a  fibrocaseous  tubercle  develops.  If  the  caseous  material 
becomes  completely  liquefied  and  absorbed  the  inflammatory  cells 
disappear  more  and  more  and  the  originally  cellular  and  partly 
caseous  tubercle  becomes  completely  replaced  by  fibrous  connective 
tissue,  and  in  place  of  the  original  cellular  tubercle  there  is  now  a 
fibrous  nodule.  As  the  tubercle  disappears  its  elements  may  be 
replaced  by  a  deposit  of  lime  salts,  and  this  process  is  termed  calcareous 
degeneration  of  the  tubercle. 

The  inflammatory  cells  of  the  tubercle  in  the  degenerative  processes 
generally  become  necrotic.  According  to  Hektoen's  observations, 
sometimes  in  healing  tuberculosis,  young  not  yet  necrotic  giant  cells 
may  break  up  into  mononuclear  cells,  and  these  as  fibroblasts  form 
connective-tissue  fibers. 

The  change  from  a  tubercle  into  a  fibrous  nodule  generally  occurs 
in  healing  tuberculosis.  Even  while  this  occurs  new  tubercles  may 
be  formed  in  the  neighborhood  and  the  process  spread.  Even  calcified 


FIBROSIS  AND  HYPERPLASTIC  TUBERCULOSIS  335 

tubercles  or  fibrous  nodules  may  still  contain  live  virulent  tubercle 
bacilli.  These  finally  disappear  entirely  or  they  remain  latent  for  a 
long  time,  and  later  lead  to  a  fresh  outbreak  of  the  tubercular  process. 
Fibrosis,  caseation,  and  calcification  may  also  continue  more  or  less 
simultaneously  in  numerous  tubercles  in  a  larger  area  and  lead  to 
the  formation  of  large  masses  of  fibrous  connective  tissue,  caseous 
or  calcereous  material. 

Cryptogenetic  Tubercular  Infection. — The  tubercle  bacillus,  as 
already  pointed  out,  generally  enters  the  body  of  man  and  the  sus- 
ceptible lower  animals  through  the  respiratory  passages  or  through  the 
gastro-intestinal  tract.  It  is  in  these  parts  and  their  lymph  glands  that 
the  tubercular  lesions  generally  first  make  their  appearance.  The 
bacillus,  however,  may  enter  the  system  through  its  usual  portals  of 
entrance  and  may  not  produce  morbid  changes  in  those  places,  but  be 
carried  away  by  the  lymph  or  blood  current  to  produce  its  first  lesions 
at  places  which  have  no  direct  open  communication  with  the  outside 
world.  Such  places  are,  for  instance,  the  bones,  the  periosteum,  and 
the  synovial  membranes  of  the  joints,  the  brain,  the  testicles,  etc. 
This  is  known  as  a  cryptogenetic  infection,  which  means  an  infection 
of  secret  or  hidden  origin.  Tubercular  infections,  however,  which  do 
not  first  involve  the  regional  lymph  glands  near  their  portal  of  entrance 
are,  on  the  whole,  very  rare. 

Fungous  Tubercular  Granulations. — The  tubercle  bacillus  may 
become  localized  from  the  beginning,  not  merely  at  a  single  point,  but 
over  a  larger  area  of  a  certain  structure.  It  may  also  rapidly  spread 
locally  and  lead  to  the  formation  of  numerous  tubercles  between 
which,  in  consequence  of  the  inflammatory  irritation,  a  great  deal 
of  ordinary  inflammatory  granulation  tissue  develops  in  which  the 
tubercles  lie  embedded.  These  soft  spongy  masses  which  develop  are 
called  fungous  tubercular  granulations.  They  are  seen  particularly 
in  the  synovial  membranes  of  the  joints  and  the  bursse. 

Solitary  Tubercles. — Sometimes  masses  of  tubercular  granulation 
tissue  form  a  single  round  or  oval  mass  of  the  size  of  a  hazel  nut  or 
walnut.  Tubercles  of  this  kind  are  called  solitary  tubercles.  They  are 
not  infrequently  seen  in  the  brain  or  its  membranes  of  man  and  cattle. 
These  round  masses,  of  course,  do  not  represent  one  tubercle,  but 
are  made  up  of  numerous  miliary  and  submiliary  tubercles. 

Fibrosis  and  Hyperplastic  Tuberculosis. — As  has  already  been  stated 
the  tubercle  may  in  healing  undergo  a  fibrosis  and  become  changed 
into  a  fibrous  nodule.  When  this  process  continues  in  a  larger  number 
of  tubercles  over  a  larger  area  it  leads  to  the  formation  of  a  consider- 
able amount  of  fibrous  connective  tissue.  It  also  occurs,  particularly 
in  the  intestines  of  man,  that  a  tubercular  process  is  comparatively 
mild  from  the  beginning  and  does  not  give  rise  to  many  cellular 
tubercles,  with  subsequent  caseation,  but  rather  leads  to  the  forma- 
tion of  a  very  large  amount  of  fibrous  connective  tissue.  This  form 
of  tuberculosis  is  known  as  a  hyperplastic  tuberculosis. 


336  TUBERCULOSIS 

Caseous  Infiltration. — When  many  tubercles  in  the  same  area 
undergo  caseation,  and  when  these  caseous  masses  become  more  or 
less  confluent  and  form  one  larger  territory,  the  process  is  termed 
caseous  infiltration  of  a  part  or  organ.  This  ^  form  of  caseous  infil- 
tration can  best  be  seen  in  the  lungs  of  cattle  before  cavity  formation 
has  occurred  and  in  the  lymph  glands.  The  tissues  of  these  structures 
become  infiltrated  by  a  rather  dry,  elastic,  cheesy  material,  generally 
grayish  white  or  light  grayish  in  man,  and  more  decidedly  yellowish 
(like  the  boiled  yolk  of  an  egg)  in  cattle.  In  the  latter  these  caseous 
infiltrations  may  lead  to  the  formation  of  masses  of  several  pounds' 
weight.  When  they  have  attained  such  a  large  size  they  are  frequently 
riddled  with  areas  where  the  caseous  material  has  been  softened  and 
even  completely  liquefied. 

Cold  Abscess  and  Cavity  Formation. — When  the  caseation  and 
liquefaction  of  neighboring  tubercles  lead  to  the  formation  of  a 
larger  abscess  filled  with  more  or  less  liquefied  material  which 
may  be  discharged  through  an  open  ulcer  or  a  fistulous  tract  the 
process  is  known  as  a  tubercular  or  a  cold  abscess.  Such  an  abscess  is 
generally  surrounded  by  a  wall,  on  the  inner  free  surface  of  which 
may  be  seen  miliary  and  submiliary  tubercles.  When  these  abscesses 
have  been  formed  in  the  lungs  and  have  discharged  part  of  their 
fluid  contents  they  are  known  as  pulmonary  cavities,  or  caverns. 
They  generally  break  by  ulceration  or  the  formation  of  a  fistulous 
tract  into  a  bronchus,  and  discharge  their  contents  by  coughing. 
Such  cavities,  as  an  invariable  rule,  sooner  or  later  become  infected 
from  the  outside  by  other  microorganisms  (staphylococci,  streptococci, 
Bacillus  pyocyaneus,  etc.).  This  process  is  known  as  a  mixed  tuber- 
cular infection.  Tubercular  lesions  of  the  intestines,  after  they  have 
ulcerated,  generally  become  infected  with  the  colon  bacillus.  In 
tuberculosis,  abscess  formation  occurs  not  only  in  soft  tissues,  but  it 
may  also  take  place  in  the  bones.  Here  the  tubercular  abscess  or 
cavity  generally  contains  bone  fragments  or  bone  sand,  and  also 
occasionally  complete  sequestra  floating  in  the  softened  tubercular 
material;  sometimes  the  area  where  the  bone  has  been  broken  down 
and  carried  away  is  filled  with  a  fungous  tubercular  granulation 
tissue. 

Tubercular  Ulcers. — Tubercular  ulcers  are  frequently  found  in  the 
intestines,  the  larynx,  trachea,  and  bronchi  as  a  result  of  tubercles 
or  confluent  tubercles  which  have  first  undergone  caseation  in  the 
centre,  the  necrotic  process  having  broken  through  the  outer  surface 
and  having  led  to  the  formation  of  open  sores.  The  latter  are 
generally  circular  or  oval,  with  a  ragged,  irregular  base,  grayish-red 
or  yellowish,  mottled,  often  studded  with  tubercles  which  surround 
them  at  the  margin. 

Miliary  Tuberculosis. — Tubercle  bacilli  may  spread  over  a  larger 
area,  for  instance  in  the  pleura,  the  peritoneum,  the  liver,  or  the 
spleen,  in'such  a  manner  as  to  form  numerous  small  tubercles,  which 


TUBERCULOSIS  IN  MAN 


337 


can  be  recognized  individually  as  nodules  of  a  somewhat  larger  or 
smaller  size  than  a  millet  seed.  This  form  is  called  a  miliary  tuber- 
culosis. It  is  generally  due  to  a  simultaneous  or  gradual  distribution 
of  tubercle  bacilli  by  the  bloodvessels  or  the  lymphatics  of  the  affected 
area  (hematogenous  or  lymphogenous  infection  of  an  area  or  organ). 
General  Acute  Miliary  Tuberculosis. — It  sometimes  occurs  that  a 
mass  of  caseous  material  breaks  into  a  large  lymph  channel  (thoracic 
duct)  or  into  a  larger  bloodvessel,  generally  a  vein,  and  that  tubercle 
bacilli  are  disseminated  in  this  manner  over  the  entire  body.  Miliary 
tubercles  are  then  formed  in  almost  all  of  the  internal  organs  of  the 
body  and  a  general  acute  miliary  tuberculosis  develops.  This  is  an 
absolutely  and  rapidly  fatal  condition  which  in  man  may  first  be 
mistaken  for  an  attack  of  typhoid  fever. 

FIG.  149 


Tuberculosis  of  the  pleura  in  cattle.     (Pearl  disease.) 

Pearl  Disease. — The  primarily  small  miliary  tubercles  usually 
grow  larger,  fuse,  and  in  this  manner  form  larger  nodules,  ranging  in 
size  from  a  pea  to  a  hazel  nut.  They  either  become  caseous,  fibrous, 
or  calcareous,  and  the  entire  pleura,  particularly  in  cattle,  may  be 
found  studded  with  them.  This  picture  of  chronic  miliary  tuber- 
culosis in  cattle  has  led  to  the  designation  "pearl  disease." 

Tuberculosis  in  Man. — Almost  every  organ  or  part  of  the  human 
body  may  be  the  seat  of  a  primary  tubercular  infection.  The  most 
common  form  of  tuberculosis  in  man  is  the  pulmonary.  Consumption, 
22 


338  TUBERCULOSIS 

phthisis^  generally  first  appears  in  the  apices  of  the  lungs.  The 
upper  respiratory  passages  are  less  frequently  affected  primarily,  but 
primary  tuberculosis  of  the  larynx  is  not  rare.  Primary  tuberculosis 
of  the  nose  is  comparatively  rare.(^The  pleura  is  often  involved  in 
phthisis,  and  it  may  be  involved  primarily^  The  lymphatic  tissue, 
including  the  tonsils,  forms  a  favorite  soil  for  the  development  of 
tuberculosis.  Children  frequently  suffer  from  a  slow  grade  of  tuber- 
culosis of  the  lymphatic  system.  ("  In  this  form  of  the  disease  tubercle 
bacilli  frequently  cannot  be  found,  or  in  small  numbers  only,  and  the 
condition  was  formerly  generally  known  as  scrofula,  or  scrofulosi^.  Of 
the  vascular  system,  the  heart  muscle  itself  is  rarely  the  seat  of  tuber- 
cular lesions;  the  pericardium  is  involved  more  frequently.  (  The 
vessels  generally  become  involved  by  the  extension  of  a  tubercle  into 
the  vessel  wall,  but  sometimes  submiliary  and  miliary  tubercles  may 
be  developed  on  the  intima  from  bacilli  which  have  been  transported 
by  the  blood  current.  The  lymphatics,  except  the  thoracic  duct,  are 
rarely  the  seat  of  tubercles/  Of  the  gastro-intestinal  tract  the  mouth 
and  pharynx  are  occasionally  the  seat  of  the  disease,  the  esophagus 
and  stomach  almost  never,  but  the  intestines  very  frequently, 
salivary  glands  and  pancreas  are  rarely  the  seat  of  tubercular  lesions, 
the  liver  and  the  spleen  very  frequently,  particularly  the  former. 
Both  the  male  and  female  genito-urinary  organs  are  frequently 
tubercular,  particularly  the  kidneys,  testicles,  and  Fallopian  tubes. 
The  adrenals  are  occasionally  affected  by  tuberculosis,  arid  then  a 
bronze  discoloration  of  the  skin  occurs.  This  condition  is  known  as 
Addison's  or  bronze  disease.  Bones  and  joints  are  frequently  affected, 
muscles,  fascia,  and  tendons  rarely,  except  by  secondary  extension  of 
advanced  tubercular  processes  in  other  structures.  When  tuberculosis 
of  the  skin  occurs  as  a  very  chronic,  slow,  ulcerating  process  on  the 
hands  or  face  it  is  known  as  lupus.  (^There  is  also  a  rare,  warty, 
granulomatous  form  of  skin  tuberculosis  called  tuberculosis  verrucosa 
cutis.  The  so-called  postmortem  tubercles  of  the  skin  of  physicians, 
veterinarians,  butchers,  etc.,  are  fairly  common.y 

Tuberculosis  in  Cattle. — The  organs  most  frequently  affected  in 
tuberculosis  of  cattle  are,  as  in  man,  the  lungs.  In  the  early  stages 
the  affection  assumes  the  character  of  a  miliary  tuberculosis  of  one  or 
more  lobes.  The  small  grayish-white  nodules  are  found  distributed 
in  a  generally  congested  pulmonary  tissue.  In  the  later  stages 
larger  nodules  are  seen  which,  when  incised,  discharge  a  dry,  caseous, 
or  a  more  liquid  material.  Caseation  by  confluence  and  agglom- 
eration of  numerous  large  and  small  tubercles  in  the  latter  stages, 
sometimes  involves  considerable  portions  of  the  lungs,  and  the  process 
is  then  known  as  a  caseous  infiltration.  Cavity  formation  in  the 
lungs  occurs  in  advanced  cases  in  cattle  as  in  man,  while  the  formation 
of  numerous  hard  fibrous,  caseous,  or  calcareous  nodules  in  the  pleura 
leads  to  that  picture  of  tuberculosis  in  cattle  called  pearl  disease. 
Tubercular  affections  of  the  abdominal  organs  and  the  gastro- 


TUBERCULOSIS  IN  CATTLE  339 

intestinal  tract  is  next  in  frequency.  The  mouth  and  tongue  rarely 
present  tubercular  ulcerations,  while  the  intestines  are  very  frequently 
affected.  The  liver  and  the  spleen  are  often  involved,  likewise  the 
organs  of  the  genito-urinary  tract,  particularly  the  kidneys,  testicles, 
uterus,  etc.  While  tuberculosis  of  the  mammary  gland  is  rare  in  man, 
it  is  common  in  cattle.  Tuberculosis  of  the  udder  generally  begins 
as  a  non-painful,  not  hot  consolidation  of  the  tissues  of  one  or  both 
posterior  ventricles  of  the  udder.  The  area  of  consolidation  spreads 
slowly,  involving  the  neighboring  ventricles  and  finally  forming 
large  (up  to  the  size  of  a  child's  head),  very  hard,  uneven,  nodular 
masses  which  push  aside  the  non-infected  parts  of  the  udder  and 
bring  about  pressure  atrophy  in  them.  The  tubercular  process  may 
also  begin  from  a  number  of  individual  nodules,  which  are  at  first 
distinct  but  later  become  confluent  and  fuse  into  each  other.  The 
regional  lymph  glands  become  enlarged  and  hardened.  Sometimes 
the  lymph  glands  above  are  enlarged  and  a  tubercular  focus  cannot  be 
detected  in  the  udder,  but  it  is,  as  a  rule,  present,  though  so  small 
that  it  does  not  become  palpable.  The  lymph  glands  in  cattle  are 
frequently  infected  in  all  forms  of  tuberculosis.  Tuberculosis  of  the 
bone  is  generally  not  primary  but  secondary  in  cattle.  Tuberculosis 
of  the  central  nervous  system  occurs  in  the  form  of  solitary  tubercles 
and  also  as  a  diffuse  cerebrospinal  tubercular  meningitis.  Acute 
miliary  tuberculosis  occurs  and  generally  kills  the  animal  in  a  very 
short  time. 

Nocard  and  Leclainche  give  the  following  figures  as  to  the  frequency 
of  the  organs  involved : 

Organs  found  affected  in  tubercular  male  cattle — 

Lungs  in  70  out  of  100  cases. 

Visceral  pleura  in  55  out  of  100  cases. 

Peritoneum  in  48  out  of  100  cases. 

Costal  pleura  in  7  out  of  100  cases. 

Liver  in  28  out  of  100  cases. 

Spleen  in  19  out  of  100  cases. 

Trachea  in  3  out  of  100  cases. 

Intestines  in  1  out  of  100  cases. 

Heart  in  0.9  out  of  100  cases. 

Kidneys  in  0.7  out  of  100  cases. 

Bone  in  0.2  out  of  100  cases. 

Larynx  in  0.15  out  of  100  cases. 

Brain  in  0.04  out  of  100  cases. 

Cord  in  0.03  out  of  100  cases. 

Tongue  in  0.01  out  of  100  cases. 
In  generalized  tuberculosis  the  organs  affected  were  found  to  be — 

Lungs  in  100  out  of  100  cases. 

Liver  in  85  out  of  100  cases. 

Intestines  in  75  out  of  100  cases. 

Serous  membranes  in  57.4  out  of  100  cases. 


340 


TUBERCULOSIS 


Kidneys  in  52.2  out  of  100  cases. 
Muscles  in  42.3  out  of  100  cases. 
Spleen  in  18.5  out  of  100  cases. 
Bone  in  8.8  out  of  100  cases. 

In  cows  the  female  genital  organs  were  found  affected  in  general 
tuberculosis — 

Uterus  in  65  out  of  100  cases. 
Udder  in  15  to  25  out  of  100  cases. 
Ovaries  in  5  out  of  100  cases. 

FIG.  150 


Tuberculosis  of  the  lung  of  a  hog. 

Tuberculosis  of  Hogs. — Since  hogs  generally  contract  tuberculosis 
by  the  ingestion  of  food  containing  tubercular  material  the  lesions 
are  most  frequently  found  in  the  gastro-intestinal  tract  and  its  lymph 
glands.  The  tubercular  affection  of  the  lymph  nodes  in  hogs  is  also 
spoken  of  as  scrofulosis.  The  glands  involved  are  generally  the 
submaxillary,  pharyngeal,  cervical,  mesenteric,  sublumbar,  and  others. 


HISTOLOGY 


341 


The  agglomeration  of  tubercular  glands  form  irregular  and  nodular 
hard,  not  painful  masses  up  to  the  size  of  a  fist.  Later  they  become 
softened,  fluctuation  followed  by  ulceration  appears,  and  the  fistulous 
tracts  formed  discharge  a  purulent,  caseous  secretion.  The  intestines 
show  infiltrations,  nodule  formation,  and  ulceration.  Tuberculosis  of 
the  liver  and  spleen  is  common  in  hogs;  the  bones  and  the  muscles 
become  secondarily  involved.  The  lungs,  the  pleura,  and  the  peri- 
toneum generally  show  the  form  of  a  miliary  tuberculosis.  Tuber- 
culosis of  the  central  nervous  system  occurs,  but  it  is  rare.  Acute 
miliary  tuberculosis  also  occurs  in  the  hog,  and  is  rapidly  fatal. 

FIG.  151 


Transverse  section  through  the  spinal  cord  of  a  hog  suffering  from  tubercular  cerebrospinal 
meningitis.     (Preparation  by  Dr.  L.  E.  Day.) 

Histology. — Stewart  and  Kinsley,  who  have  studied  the  histology  of 
the  tubercle  in  hogs,  found  that  there  are  two  general  types.  One 
type  is  represented  by  the  cellular  necrotic  and  calcified  necrotic 
tubercle.  "This  group  of  tubercular  lesions  is  probably  the  result  of  the 
activity  of  quite  virulent  tubercle  bacilli.  The  second  type  of  lesions 
are  those  described  as  fibrous  tubercles,  which  may  be  preceded  by 
cellular  tubercles  and  usually  contain  calcareous  foci  in  the  later 
stages;  these  lesions  are  no  doubt  the  result  of  infection  with  slightly 
virulent  tubercle  bacilli.  In  the  examination  of  770  sections  no  giant 
cells  were  seen,  and  it  is,  therefore,  believed  that  the  tissue  reaction  in 
hogs  against  infection  with  the  tubercle  bacillus  does  not  favor  the 
production  of  giant  cells." 


342  TUBERCULOSIS 

While  this  statement  is  undoubtedly  true,  giant  cells  are  seen  in 
some  tubercular  lesions  in  the  hog.  The  author  is  indebted  to  Dr. 
Enos  L.  Day  for  sections  of  a  case  of  tuberculosis  of  the  skin  in  a  hog. 
These  sections  show  a  number  of  giant  cells;  they  are,  however,  not 
very  numerous.  Giant  cells  are  also  occasionally  found  in  other 
tubercular  lesions  of  swine. 

Fia.  152 


Tuberculosis  of  the  bronchial  glands  of  a  hog. 

Tuberculosis  in  Other  Mammals. — Tuberculosis  in  goats  and  sheep 
is  very  rare.  Tuberculosis  in  dogs  and  cats  occurs  not  infrequently 
when  these  animals  are  living  with  tubercular  persons  and  when  they 
inhale  pulverized  sputum  or  ingest  food  contaminated  with  it.  The 
most  common  form  of  tuberculosis  in  dogs  or  cats  is  that  of  the  lungs 
or  of  the  intestines. 

Tuberculosis  in  the  horse  is  not  very  common,  though  the  animal 
is  quite  susceptible  to  artificial  inoculation  with  pure  cultures  of 
tubercle  bacilli  or  with  tubercular  material;  but  it  appears  that  the 


AVIAN  TUBERCULOSIS  343 

more  healthy  outdoor  life  with  exercise  which  this  animal  generally 
leads  makes  it  much  less  susceptible  to  the  natural  infection  than 
man,  cattle,  or  swine.  When  tuberculosis  is  encountered  in  the  horse 
it  is  generally  found  in  young  animals  in  which  the  mesenteric  and 
other  abdominal  glands  are  the  favorite  seat  of  infection.  These 
glands  are  found  enlarged  and  form  agglomerated  nodular  tumor 
masses;  the  mesentery  and  omentum  are  thickened  and  the  intestinal 
mucous  membrane  often  shows  tuberqular  ulcers.  The  tubercular 
masses  may  surround  large  veins,  compress  and  discharge  into  them 
tubercular  material,  bringing  about  a  general  acute  miliary  tuber- 
culosis. In  abdominal  tuberculosis  in  the  horse  the  spleen  is  frequently 
found  involved,  the  liver  rarely.  The  former  may  assume  a  large  size 
and  may  weigh  twenty  to  twenty-five  pounds.  Primary  pulmonary 
tuberculosis  in  the  horse  generally  is  of  the  miliary  type;  the  large 
tubercular  nodules  and  masses  seen  in  cattle  rarely  occur;  the  peri- 
bronchial  lymph  nodes  are  found  enlarged  and  studded  with  yellowish 
nodules,  while  the  respiratory  mucosa  is  sometimes  ulcerated.  When 
the  serous  membranes,  the  pleura,  and  the  peritoneum  are  the  seat  of 
tubercular  lesions  in  the  horse,  they  present  a  picture  similar  to  pearl 
disease  in  cattle. 

Tuberculosis  in  Food-producing  Animals  in  the  United  States. — Melvin, 
in  a  paper  on  the  Economic  Importance  of  Tuberculosis  of  Food- 
producing  Animals,  read  before  the  Sixth  International  Congress  on 
Tuberculosis,  held  in  Washington  in  1908,  reported  that  the  following 
number  of  animals  were  found  tubercular  among  a  total  of  53,973,337 
head  slaughtered  in  the  United  States,  under  federal  inspection,  during 
the  fiscal  year  1907-08: 

Tubercular  cattle 68,395 

Tubercular  calves '     ;      .      .      .      .      .      .  524 

Tubercular  hogs 719,300 

Tubercular  sheep ., 40 

Tubercular  goats .'..»•.'    •".'"..  1 

Loss  for  condemned  cattle,  $710,677;  hogs,  $1,401,723.  The  same 
author  estimates  the  loss  due  to  tuberculosis  among  meat-  and  milk- 
producing  animals  in  the  United  States  at  $14,000,000  annually. 

Avian  Tuberculosis. — Robert  Koch,  in  1882,  was  of  the  opinion 
that  avian  and  mammalian  tuberculosis  were  the  same  disease  and 
due  to  an  identical  microorganism.  Later,  however,  he  withdrew 
from  this  belief,  and  the  preponderance  of  evidence  today  is  that  these 
two  types  of  tuberculosis  are  not  absolutely  identical,  but  that  they 
are  in  some  important  respects  dissimilar  diseases,  due  to  two  different 
varieties  of  the  tubercle  bacillus.  Avian  tuberculosis  occurs  among 
chickens  and  pigeons,  also  occasionally  among  geese  and  ducks. 
Natural  infection  occurs  when  healthy  animals  feed  upon  material 
soiled  with  the  discharges  of  tubercular  birds  or  when  they  directly 
eat  some  of  the  tubercular  organs.  Tuberculosis  in  birds  most 
commonly  occurs  in  the  abdominal  organs  and  the  intestines  and 


344  TUBERCULOSIS 

the  bacilli  are  spread  through  the  feces.  The  liver  and  spleen  are 
usually  affected  even  if  the  intestines  do  not  show  any  tubercular 
lesions.  Frequently,  however,  the  first  pathologic  changes  are  found 
in  the  intestines.  Here  the  bacilli  enter  the  mucosa,  where  they 
produce  tubercles  which  soon  degenerate  and  form  ulcers.  From 
the  intestines  the  bacilli,  through  the  blood  current,  enter  the  liver 
and  later  the  spleen,  lungs,  joints  and  fascia?  of  the  tendons.  The 
livers  in  birds  dead  of  tuberculosis  are  generally  much  enlarged;  the 
parenchyma  cells  may  show  a  marked  degree  of  fatty  degeneration. 
The  numerous  tubercles  vary  in  size  from  a  millet  seed  to  a  pea  and 
even  larger.  These  nodules  contain  a  dry,  yellowish,  grumous, 
caseous,  sometimes  chalky  material.  The  mucosa  of  the  intestine, 
when  affected,  shows  yellow  nodules  and  funnel-shaped  tubercular 
ulcers.  The  mesentery  may  show  larger  nodules.  The  tubercles  in 
birds  differ  somewhat  from  those  in  mammals,  because  the  degen- 
eration leads  to  the  formation  of  a  material  which  is  less  granular  and 
more  hyaline  and  dryer  than  that  found  in  mammalian  tuberculosis. 
Nocard  and  Leclainche  describe  the  degenerated  material  in  avian 
tuberculosis  as  a  detritus  with  hyaline  blocks,  both  infiltrated  by  an 
amyloid  material.  Such  amyloid  material  is  also  found,  according  to 
Kitt,  infiltrating  the  intestinal  wall.  Tubercles  in  ordinary  fowls 
generally  do  not  show  many  giant  cells,  few  lymphoid  cells,  and 
mostly  epithelioid  cells  and  fibroblasts;  but  there  appear  to  be  very 
numerous  giant  cells  in  tuberculosis  of  guinea-fowl  (Numida  meleag- 
ris).  In  tuberculosis  of  the  liver  in  these  birds,  as  observed  by  the 
author,  the  giant  cells  are  either  round  or  oval,  or  they  form  irregular, 
apparently  syncytial  masses  with  nuclei  irregularly  grouped  in  the 
middle.  Tubercular  lesions  are  also  frequently  seen  in  fowl  in  the 
abdominal  lymph  glands  and  on  the  visceral  layer  of  the  peritoneum. 
Joint  and  bone  tuberculosis  are  also  common  and  lead  to  the  formation 
of  considerable  dry  caseous  masses;  the  cartilages  and  the  epiphyses 
are  thickened  and  corroded  in  joint  tuberculosis.  Tuberculosis  of 
the  lungs  is  less  frequent  in  fowl  than  tubercular  affections  of  the 
organs  named  above.  Tubercle  bacilli  are  generally  found  in  large 
numbers  in  the  tubercular  lesions  of  fowl. 

Tuberculosis  of  Parrots. — While  quite  frequently  observed,  this 
is  not  a  true  avian  tuberculosis,  but  an  infection  of  these  birds  with 
human  tubercular  material.  They  generally  contract  the  disease 
directly  or  indirectly  from  the  sputum  of  consumptive  persons.  The 
portal  of  entrance  of  the  virus  infecting  parrots  is  generally  found 
in  the  skin  of  the  head  or  the  mucosa  of  the  mouth,  nose,  or  eyes. 
In  advanced  cases  the  abdominal  organs,  the  joints,  and  bones  are 
found  affected,  as  in  chickens  in  true  avian  tuberculosis. 

Fish  and  Turtles. — A  pathologic  change  with  the  formation  of  nodules 
due  to  the  presence  of  acid-fast  bacilli  has  also  been  described  in  fish 
and  turtles. 


PLATE   IX 


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Avian  Tuberculosis. 

Section  of  the  liver  of  a  guinea-fowl. 


QUESTIONS  345 


QUESTIONS. 

1.  What  is  the  characteristic   anatomic  lesion  of  tuberculosis?      How  can 
its  formation  best  be  studied,  and  why? 

2.  Describe  the  formation  of  an  experimental  tubercle. 

3.  Describe  the  formation  of  a  polynuclear  giant  cell.    What  is  an  epithelioid 
cell?    What  is  a  lymphoid  cell? 

4.  Describe  the  cellular  elements  of  a  young  tubercle  and  their  arrangement 
in  the  nodule. 

5.  Describe  the  new  formation  of  vessels  in  the  tubercle. 

6.  What  is  the  evidence  which  shows  that  there  occurs  rapid  cell  proliferation 
after  the  entrance  of  virulent  tubercle  bacilli  into  the  tissues  of  a  susceptible 
animal  ? 

7.  Describe  the  relation  between  a  multinuctear  giant  cell  and  the  tubercle 
bacilli  contained  in  it. 

8.  Does  the  tubercle  contain  fibrous  connective  tissue?    What  is  a  fibrous 
tubercle  ? 

9.  Does  a  tubercle  contain  polynuclear  leukocytes,  and  if  so,  what  is  their 
mode  of  entrance? 

10.  What  is  meant  by  the  caseation  of  the  tubercle?    What  kind  of  process 
is  caseation? 

11.  What  is  a  fibrinous  substance?    To  what  factors  are  the  peculiar  physical 
properties  of  caseous  material  attributed? 

12.  What  secondary  changes  occur  in  the  caseous  tubercle?  to  what  are  they 
due? 

13.  Describe  fungous  tubercular  granulations. 

14.  What  is  a  fibrocaseous  tubercle? 

15.  What  may  be  the  fate  of  the  giant  cells  in  healing  tuberculosis? 

16.  What  is  a  solitary  tubercle  ?    Where  are  they  generally  found  ? 

17.  What  is  a  hyperplastic  tuberculosis? 

18.  What  is  meant  by  caseous  infiltration?    Where  generally  found? 

19.  What  is  a  cold  abscess?    What  a  tubercular  cavity?    Describe  its  wall. 

20.  What  is  a  mixed  tubercular  infection? 

21.  Describe  a  tubercular  ulcer. 

22.  What  is  a  miliary  tuberculosis?    How  is  acute,  general  miliary  tuberculosis 
commonly,  if  not  always,  brought  about  ? 

23.  What  is  the  meaning  of  the  term  pearl  disease?    Why  so  called? 

24.  Give  in  general  outlines  the  organs  affected  by  tuberculosis  in  man. 

25.  Name  the  organs  most  generally  affected  in  cattle,  and  describe  the  patho- 
logic lesions  as  most  commonly  found  in  cattle. 

26.  Discuss  tuberculosis  in  sheep,  goats,  cats,  dogs,  and  horses 

27.  Describe  tubercular  lesions  in  chickens  and  pigeons.    How  is  the  disease 
generally  spread  among  these  domestic  fowls? 

28.  What  kind  of  tuberculosis  do  parrots  generally  contract? 


CHAPTER    XXX. 

THE    BACILLUS    OF  TUBERCULOSIS— TUBERCULIN  TESTS— BOVO- 

VACCINE— THE  INTERTRANSMISSIBILITY  OF  BOVINE  AND 

HUMAN  TUBERCULOSIS— AVIAN  TUBERCULOSIS. 

Morphology. — The  tubercle  bacillus  is  a  rod-shaped  organism, 
generally  rather  slender  when  found  in  human  tuberculosis,  more 
plump  in  the  bovine  type,1  and  commonly  even  more  coarse  in  the 
avian  type.  It  measures  from  1.5  to  3.5  micra  in  length  and  0.2  to 
0.5  micron  in  breadth.  It  frequently  occurs  in  pairs,  the  two  individ- 
uals may  or  may  not  be  in  direct  contact.  When  bacilli  are  numerous 
in  sputum,  pus,  urine,  or  other  fluid  or  semifluid  tubercular  products 
they  often  present  themselves  in  parallel  groups  or  clusters.  The 
bacilli  often  have  a  beaded,  granular  appearance  which  is  at  times  so 
pronounced  that  beginners  gain  the  impression  that  they  are  dealing 
with  short  chains  of  streptococci.  Under  some  conditions  branching 
forms  are  seen;  this  proves  that  the  organism  of  tuberculosis  is  perhaps 
not  a  bacillus,  but  more  properly  a  member  of  the  streptothricse  to 
which  the  ray  fungus  or  actinomyces  belongs.  The  bacilli  frequently 
show  in  their  interior  unstained,  oval,  or  slightly  biconcave  spaces, 
which  were  formerly  mistaken  for  spores.  It  is  now  unanimously 
agreed  that  the  tubercle  bacillus  does  not  form  spores,  because  the 
forms  which  show  these  unstained  spaces  are  not  more  resistant  than 
the  uniformly  stained  bacilli.  This  fact  indicates  the  absence  of  the 
most  important  characteristics  of  the  true  spore — namely,  its  greater 
resistance  to  antiseptics,  higher  temperatures,  drying  out  processes, 
etc.  The  tubercle  bacillus  is  not  motile  and  does  not  possess  flagella. 
It  does,  however,  possess  a  capsule  containing  a  wax-like  substance 
to  which  the  bacillus  owes  two  remarkable  features — namely,  its 
peculiar  staining  properties  and  its  resistance,  which  is  greater  than 
that  of  most  other  non-sporulating  pathogenic  bacteria.  It  is  also 
more  resistant  to  drying  out  than  most  of  the  latter. 

Staining  Properties. — On  account  of  the  tough,  tenacious  wax- 
containing  capsule,  which  surrounds  the  tubercle  bacillus,  it  cannot, 
like  most  other  pathogenic  bacteria,  be  stained  with  the  ordinary 
watery  anilin  stains.  It  requires  a  more  intense  stain,  which  either 
must  act  for  a  considerable  time  or  be  heated  to  intensify  its  action. 
After  once  having  taken  the  stain,  however,  the  tubercle  bacillus 
holds  it  very  firmly  against  the  decolorizing  action  of  dilute  acids, 

1  The  difference  between  the  human  and  bovine  types  of  the  tubercle  bacillus  will  be  considered 
more  fully  under  the  discussion  of  the  intertransmissibility  of  human  and  bovine  tuberculosis. 


PLATE  X 


Smear  from  a  Tubercular  Spleen  of  Cattle. 
Zeiss  horn,  oil  immersion  objective  2  mm.    Compens.  ocular  No.  6. 


STAINING  PROPERTIES  347 

alcohol,  etc.  If,  therefore,  a  pathologic  product  containing  tubercle 
bacilli  and  other  bacteria  is  treated  with  an  intense  hot  staining  solution 
and  then  subjected  to  the  action  of  a  decolorizing  fluid  and  afterward 
counterstained  with  a  contrast  stain,  the  tubercle  bacilli  will  have 
kept  the  first  stain,  while  other  bacteria  have  been  dyed  with  the 
counterstain.  In  making  use  of  this  principle,  tubercle  bacilli  can 
easily  be  stained  and  distinguished  from  other  bacteria.  There  are, 
however,  a  few  bacilli  which  act  in  a  manner  similar  to  the  tubercle 
organisms  These  and  the  methods  for  their  differentiation  will  be 
considered  below.  The  bacilli  of  this  group  are  known  as  acid-fast 
bacilli. 

STAINING  TUBERCLE  BACILLI  IN  FLUID  AND  SEMIFLUID  MATERIAL. 
— Reagents  Required: 

(a)  Ziehl's  Carbol-fuchsin. 
1    Carbolic  acid,  5  c.c. 

2.  Distilled  water,  100  c.c. 

3.  Basic  fuchsin  crystals,  1  gram. 

4.  Absolute  alcohol,  10  c.c. 
Dissolve  1  in  2  and  3  in  4  and  mix. 

(6)  10  per  cent,  watery  solution  of  nitric  acid. 

(c)  95  per  cent,  alcohol. 

(d)  Ordinary  watery  solution  of  methylene  blue. 
Steps  in  Staining,  Decolorizing,  Counter  staining. — 

1.  Prepare    a    cover-glass    from    suspected    material,    preferably 
selecting  some  of  the  cheesy  flocculi,  as  they  are  found  in  sputum, 
pus,  etc.      After  such  material  has  been  spread  on  a  cover-glass, 
allow  it  to  become  air  dry,  and  fix  by  passing  three  times  through  a 
flame. 

2.  Apply  to  the  cover-glass,  held  in  a  suitable  forceps,  enough  of 
Ziehl's  carbol-fuchsin  so  that  the  whole  surface  is  well  covered;  now 
hold  high  over  a  small  flame  until  the  staining  solution  boils.    Then 
set  it  aside  for  a  few  minutes  to  permit  the  hot  staining  solution  to 
act  well. 

3.  Pour  off  the  hot  staining  solution  and  wash  the  cover-glass  in 
ordinary  tap  water  or  in  distilled  water. 

4.  Dip  rapidly  into  the  10  per  cent,  watery  nitric-acid  solution, 
and  at  once  wash  freely  in  95  per  cent,  alcohol.     Continue  washing 
the  cover-glass,  which  is  held  in  the  forceps,  in  alcohol  until  apparently 
all  of  the  red  stain  has  been  washed  out,  i.  e.,  until  the  cover-glass 
again  appears  almost  unstained. 

5.  Dry  between  filter  paper  and  for  three  or  four  minutes  apply  a 
thin  watery  solution  of  methylene  blue. 

6.  Wash  in  water,  dry  between  filter  papers,  mount  on  a  slide  in 
the  usual  manner. 

Result  of  the  Procedure  Employed. — The  tubercle  bacilli  are  stained 
red;  all  other  bacteria,  yeast  cells,  etc.,  and  cell  nuclei  are  stained 
blue. 


348  THE  BACILLUS  OF  TUBERCULOSIS 

Precaution. — The  procedure  of  staining  tubercle  bacilli  and  finding 
them  subsequently,  if  they  are  present,  is  very  easy,  provided  it  is 
done  carefully  and  a  few  precautions  are  observed.  In  the  first  place 
it  is  well  to  pour  the  suspected  sputum  or  pus  into  a  flat  vessel,  for 
instance  a  Petri  dish,  and  to  place  it  on  a  dark  background.  This 
makes  it  possible  more  easily  to  pick  out  with  the  platinum  loop  the 
small  flocculent  tubercular  masses  often  found  in  tubercular  material. 
In  heating  the  carbol-fuchsin  solution  to  boiling  it  must  not  be  allowed 
to  evaporate  down  too  much,  because  then  precipitates  may  be  formed 
on  the  cover-glass.  After  having  poured  the  hot  stain  off  the  cover- 
glass,  washing  in  water  is  necessary,  because  if  it  is  dipped  directly 
into  the  dilute  nitric  acid  the  latter  and  the  carbolic  acid  of  the  stain 
may  form  a  smeary  substance  on  its  surface.  The  cover-glass  must 
never  be  allowed  to  remain  in  the  10  per  cent,  nitric-acid  solution 
long;  it  should  just  receive  a  dip.  If  after  prolonged  washing  in 
alcohol,  however,  a  good  deal  of  red  remains  in  the  cover-glass 
preparation  a  second  dip  in  the  dilute  nitric  acid  solution  is  not  only 
permissible,  but  indicated.  The  watery  methylene-blue  solution 
should  not  be  too  concentrated,  nor  should  it  act  too  long,  because  if 
it  does  and  there  is  much  cellular  material  in  the  preparation  an 
intense  blue  stain  may  cover  up  a  small  number  of  red-stained  tubercle 
bacilli.  When  staining  for  tubercle  bacilli  in  perfectly  fluid  media, 
such  as,  for  instance,  urine,  the  fluid  is  first  centrifuged,  the  super- 
natant clear  liquid  decanted  off,  and  the  cover-glass  preparation  made 
from  the  sediment,  which  is  then  treated,  stained,  decolorized,  etc., 
in  the  usual  manner. 

Biedert's  Sedimentation  Method. — When  tubercle  bacilli  have  not 
been  found  in  suspected  sputum  or  tenacious  caseous  material  the 
material  may  be  liquefied  in  order  to  centrifuge  it  and  to  increase  the 
chances  of  finding  the  bacilli  if  only  a  few  are  present.  The  method 
then  best  used  is  Biedert's  sedimentation  method;  its  steps  are  as 
follows : 

1.  Place  about  a  tablespoonful  of  the  suspected  material  in  a 
porcelain  evaporating  dish  and  add  twice  the  amount  of  distilled 
water.  Mix  well  by  prolonged  stirring  with  a  glass  rod. 
,  2.  Place  the  evaporating  dish  over  a  small  flame  and  heat  to 
boiling;  continue  stirring  constantly,  and  while  doing  so  add  gradually 
ten  drops  of  a  10  per  cent,  watery  solution  of  caustic  potash.  Continue 
stirring  and  boiling  until  the  mixture  has  become  entirely  fluid  and 
has  lost  its  tenacious,  stringy  character. 

3.  Cool  and  centrifuge;    pipette  off   the  supernatant    fluid   and 
preserve  the  sediment. 

4.  Take  some  of   the  original  untreated  material  and  spread  on 
a  cover-glass;  then  add  some  of  the  sediment  and  rub  it  up  well  with 
the  material  on  the  cover-glass. 

5.  Air  dry,  fix  and  stain,  decolorize,  etc.,  as  usual. 

This  method  often  makes  it  possible  to  find  few  tubercle  bacilli 


STAINING  PROPERTIES  349 

which  have  escaped  the  ordinary  procedure  of  making  one  or  more 
cover-glass  preparations. 

STAINING  TUBERCLE  BACILLI  IN  PARAFFIN  SECTIONS  — 1.  Sections 
cut  as  thin  as  possible  are  placed  .on  clean  slides  with  Meyer's  egg- 
albumen  mixture;  they  are  then  slightly  heated  over  a  small  flame 
to  melt  the  paraffin  and  to  coagulate  the  fixing  albumen  Place 
directly  in  xylol  to  dissolve  out  the  paraffin  and  then  wash  out  the 
xylol  in  absolute  alcohol 

•  2.  Place  in  a  beaker  containing  Ziehl's  carbol-fuchsin,  and  heat 
over  a  small  flame  until  the  staining  solution  begins  to  steam  (do  not 
heat  to  boiling,  as  this  may  damage  the  tissue).  Leave  in  the  hot 
staining  solution  for  ten  or  fifteen  minutes. 

3.  Wash  well  in  water,  then  dip  rapidly  into  a  20  per  cent,  watery 
solution  of  nitric  acid,  and  immediately  wash  freely  in  strong  (95  per 
cent.)  alcohol  until  all  the  red  color  has  been  removed  from  the 
section.      A  second  dip  into  20  per  cent,  nitric-acid   solution  and 
another  washing  in  the  alcohol  may  be  necessary. 

4.  Wash  in  water  and  stain  for  five  minutes  in  dilute  Loeffler's 
alkaline  methylene  blue  (1  part  of  the  stain  to  2  parts  of  distilled 
water). 

5.  Decolorize  by  washing  first  in  95  per  cent,   alcohol,  then  in 
absolute  alcohol  until  most  of   the  blue  stain  has  been  washed  out 
again,  i.  e.,  until  the  nuclei  only  have  retained  the  blue  stain. 

6.  Clear  in  xylol,  dry  with  filter  paper. 

7.  Mount  in  Canada  balsam. 

In  order  to  obtain  a  good,  clear,  nuclear  stain,  and  to  remove  the 
excess  of  blue  properly,  it  is  necessary  to  look  with  a  low-power  lens  at 
the  slide  after  it  has  been  in  the  xylol  and  before  it  is  dried  and 
covered  with  Canada  balsam.  The  slide  still  wet  with  xylol  is  placed 
on  the  stage  of  the  microscope  and  studied.  If  the  section  is  still 
too  blue,  and  if  the  nuclei  have  not  been  well  differentiated,  the  slide 
must  be  returned  to  the  absolute  alcohol  and  again  freely  washed 
until  a  repeated  microscopic  examination  shows  that  the  desired 
effect  has  been  obtained;  then  it  may  be  mounted  permanently  and 
be  examined  with  oil-immersion  magnification  for  the  red-stained 
tubercle  bacilli. 

STAINING  TUBERCLE  BACILLI  IN  CELLOIDIN  SECTIONS. — 1.  Sections 
t  as  thin  as  possible  are  stained  rather  lightly  for  three  to  four 
minutes  in  alum  hematoxylin. 

2.  Wash  in  water. 

3.  Leave  in  warm  (not  hot)  carbol-fuchsin  solution  for  twenty  to 
thirty  minutes. 

4.  Wash  in  water. 

.    5.  Decolorize  in  acid  alcohol1  one-half  to  one  minute. 
6.  Wash  in  several  changes  of  water  to  remove  every  trace  of  acid 
that  the  hematoxylin  stain  later  shows  a  bluish  color  again. 

1  Acid  alcohol  is  composed  of  hydrochloric  acid,  1  c.c.,  and  70  per  cent,  alcohol,  99  c.c. 


350  THE  BACILLUS  OF  TUBERCULOSIS 

7.  Wash  well  in  95  per  cent,  alcohol  until  all  fuchsin  has  been 
dissolved  out.    This  may  require  several  changes  of  fresh  alcohol. 

8.  Anilin  oil  one-half  minute. 

9.  Several  changes  of  xylol. 

10.  Mount  on  a  slide  in  Canada  balsam. 

Cultural  Properties. — The  tubercle  bacillus  cannot  be  readily 
cultivated  directly  from  sputum  or  caseous  or  purulent  discharges 
containing  it  because  it  is  not  present  alone,  but  mixed  with  other 
bacteria.  Since  the  tubercle  bacillus  grows  very  slowly  in  artificial 
culture  media  the  other  bacteria  multiply  much  more  rapidly,  and, 
in  fact,  overgrow  it,  making  it  difficult  if  not  impossible  to  find  it.  It 
is  certain  that  it  can  hardly  ever  be  obtained  in  pure  culture  by 
the  ordinary  method  of  inoculating  tubes  directly  or  pouring  plates. 
The  usual  method,  therefore,  of  obtaining  pure  cultures  ^jnsists  in 
inoculating  the  tubercular  material  either  subcutaneoiftlv  or  intra- 
peritoneally  into  guinea-pigs.  This  highly  susceptible  animal  develops 
first  a  local  tuberculosis  of  the  regional  lymph  glands  and  later  a 
general  tuberculosis.  If  such  an  infected  guinea-pig  is  killed  eight 
to  ten  weeks  after  the  infection  the  proper  tubercular  material  for 
obtaining  pure  cultures  can  be  procured. 

The  procedure  is  best  carried  out  as  follows:  The  animal  is 
chloroformed  to  death  and  then  stretched  out  on  a  board  with  its 
four  legs  tied  to  four  nails.  The  abdomen  is  shaved  and  sterilized 
and  a  median  incision  is  made  from  the  sternum  to  the  symphysis. 
This  is  done  with  a  sterile  knife  and  in  such  a  manner  that  the  peri- 
toneum is  left  unopened.  The  skin  is  then  flapped  back  on  both  sides. 
The  peritoneum  is  now  opened  in  the  middle  line  with  fresh  sterile 
instruments  (scissors  or  knife)  and  held  open  with  sterile  forceps  on 
both  sides  by  an  assistant  or  fastened  to  the  side  with  suitable  tacks. 
The  operator  now  removes,  with  sterile  forceps  and  scissors,  some 
tubercular  abdominal  glands  and  the  whole  or  part  of  the  spleen. 
These  tissues  P  ^mediately  placed  in  a  covered  sterile  glass  dish 
(a  not  too  .si  .  Petri  dish  will  answer  very  well).  The  material 
so  obtained  is  then  divided,  with  all  aseptic  precautions,  into  small 
pieces,  and  portions  from  the  interior  of  tubercular  glands,  or  from 
tubercles  in  the  spleen,  are  brought  into  tubes  containing  the  proper 
culture  media,  where  they  are  left  either  undisturbed  on  the  surface 
or  rubbed  over  it  with  a  strong  platinum  loop.  Suitable  media  for 
cultivating  the  tubercle  bacillus  are  sterile  coagulated  cattle-blood 
serum  (Koch),  dog's  blood  serum  obtained  sterilly  (Smith),  eggs  in 
which  the  white  and  yellow  have  been  mixed  and  the  mass  subse- 
quently distributed  to  test-tubes  (Dorset),  and  various  other  media. 
After  inoculation  the  tubes  must  be  protected  against  evaporation,  as 
it  is  necessary  to  keep  them  in  the  incubator  at  blood  temperature  for 
several  weeks.  This  is  done  either  with  rubber  caps,  ground-glass 
stoppers,  or  by  closing  them  with  paraffin.  If  there  is  enough  moisture 
in  the  tubes  from  the  beginning  the  microorganism  will  grow  without 


RESISTANCE  351 

air-tight  closure  of  the  tube.     The  growth,  however,  is  very  slow 

when  the  culture  media  have  been  inoculated  from  infected  animals. 

In  later  successively  transplanted  generations  the  growth  is  somewhat 

more  rapid  and  the  bacilli  are  not  so  selective  as  to  the  medium  in 

which  they  grow;  after  the  first  generation,  for  instance,  they  grow 

well  on  glycerin  agar.     In  the  first  generation,  growth  generally  is  not 

easily  recognizable  with  the  naked  eye  until  ten  to  fourteen  days  after 

inoculation.    The  multiplying  bacteria  form  a  dry,  lusterless,  granular, 

more  or  less  wrinkled  growth,  slightly  yellowish  in  color.    Growth  takes 

I   place  best  at  blood  temperature,  and  does  not  occur  below  30°  C.  or 

1  above  42°  C.     The  tubercle  bacillus  is  a  strict  parasite,  which  does 

I  not  find  in  nature  the  conditions  to  grow  as  a  saprophyte.    Tubercle 

I  bacilli  have  been  found  in  the  external  world  only  where  they  have 

I  been  sprea^  bv  the  discharges  of  tubercular  persons  or  animals. 

Toxic  Effects. — These  depend  upon  two  principal  factors,  namely, 

(1)  the  metabolic  products  of  the  organism  formed  during  its  growth 
and  multiplication  in  the  invaded  tissues  of  susceptible  animals,  and 

(2)  the  poisonous  substances  contained  in  the  body  of  the  bacterium. 
The  latter  substances,  even  after  the  bacillus  is  dead  and  can  no 

I  longer  multiply,  cause  local  abscesses,  necrosis,  caseation,  marasmus, 
I  and  elevation  of  temperature.  As  has  been  shown  by  a  number  of 
|  authors,  including  Maffucci,  Prudden,  Hodenpyle,  Strauss,  and 
|  Gamalia,  dead  bacilli  injected  in  small  but  sufficient  quantities  may 
*v  produce  tubercles  with  giant  cells,  but  the  process  remains  localized 
I  and  tends  to  heal. 

Resistance. — Tubercle  bacilli  in  moist  material  are  probably  soon 
I  destroyed  by  the  putrefactive  bacteria  and  the  changes  they  bring 
£  about.  This  was,  at  least,  the  view  generally  held  formerly,  but 
|  there  are  now  on  record  some  observations  which  show  that  this  is 
5"  not  invariably  the  case.  If  tubercular  material  is  placed  in  water  the 
«..  tubercle  bacillus  may  remain  alive  for  a  considerable  time.  The 
[  bacillus  is  very  resistant  to  drying  out,  and  may  r  'n  alive  a  long 
I  time  in  desiccated  tubercular  material,  such  as  sx  ,_p**s,  and 

'*  other  discharges;  the  average  is  about  three  months.    Direct  sunlight 
|  kills  the  organism  if  it  is  spread  out  in  a  thin  layer,  within  a  few 
I  minutes,  but  diffuse  daylight  requires  five  to  seven  days.     Strauss 
found  that  glycerin  bouillon  cultures  with  an  abundant  growth  were 
J.  killed  if  exposed  to  direct  sunlight  for  two  hours     Severe  cold  has  no 
effect  upon  the  bacillus.     According  to  the  figures  given  by  Cornet 
and  Meyer  in  their  summary,  and  obtained  from  a  resume  of  the  con- 
•  siderable  literature  upon  the  subject,  the  following  periods  of  time 
and  temperature  exposures  kill  the  tubercle  bacillus  in  the  moist  state: 
Four  to  six  hours  heated  at  55°  C. 
One  hour  heated  at  60°  C. 
Ten  to  twenty  minutes  heated  at  70°  C. 
Five  minutes  heated  at  80°  C. 
One  to  two  minutes  heated  at  90°  to  95°  C. 


352  THE  BACILLUS  OF  TUBERCULOSIS 

In  order  safely  to  kill  all  tubercle  bacilli  contained  in  tubercular 
sputum  it  is  necessary  to  boil  it  for  five  minutes  at  100°  C.  The 
bacilli  are  very  resistant  to  dry  heat  and  can  withstand  100°  C.  for 
one  hour.  Corrosive  sublimate  cannot  be  safely  used  to  disinfect 
sputum,  pus,  caseous  material,  etc.,  because  a  peripheral  coagulation 
of  the  albumen  prevents  its  penetration  into  the  interior  of  the  masses 
containing  the  bacilli.  Carbolic  acid  (5  per  cent.)  added  to  tubercular 
material  in  liberal  quantities  kills  the  organism  within  a  few  hours. 
Formalin  is  not  trustworthy  as  a  disinfectant  for  tubercular  albumin- 
ous material. 

Tubercle  Bacilli  in  the  Circulating  Blood. — In  much  advanced  cases 
tubercle  bacilli  have  occasionally  been  found  in  the  circulating 
blood,  but  this  is  an  exceptional  occurrence.  They  evidently  do  not 
multiply  in  the  blood  and  are  filtered  out  very  soon  after  entering  it. 
It  therefore  occasioned  considerable  surprise  when  Rosenberger 
claimed  that  he  had  been  able  to  show  that  tubercle  bacilli  are  present 
in  the  blood  even  in  the  most  incipient  cases.  In  other  words, 
Rosenberger  claimed  that  tuberculosis  was  a  bacteriemia.  However,  it 
has  been  shown  by  McFarland  and  his  co-workers,  and  by  Ravenel 
and  Smith,  that  this  statement  is  incorrect  for  human  tuberculosis. 
Schroeder,  Col  ton,  and  Mohler  examined  the  blood  of  50  tubercular 
cows,  not  merely  by  staining  methods,  but  by  the  inoculation  of  135 
guinea-pigs,  and  found  that  Rosenberger's  claim  was  entirely  un- 
substantiated; tuberculosis  accordingly  is  a  bacteriemia  neither  in 
man  nor  cattle.  Rosenberger  in  his  painstaking  work  became  the 
victim  of  a  deceptive  acid-fast  bacillus  often  found  in  distilled  water 
used  in  laboratories. 


TUBERCULIN   TESTS— THE    DIAGNOSIS   OF   LATENT   TUBER- 
CULOSIS IN  CATTLE. 

Koch's  Old  Tuberculin.— Koch's  old  tuberculin,  also  known  as 
Tuberculin  Original,  or  Koch's  O.  T.,  is  prepared  as  follows:  A 
veal  bouillon,  containing  3  to  5  per  cent,  glycerin  and  the  usual 
amounts  of  common  salt  (J  per  cent.)  and  pepton  (1  per  cent.), 
having  been  made  slightly  alkaline  and  kept  in  a  flask,  is  inoculated 
on  the  surface  from  a  pure  culture  of  tubercle  bacilli.  The  flask  is 
then  kept  in  the  incubator  at  blood  temperature  from  three  to  six 
weeks.  During  this  time  a  thick,  dry,  crumpled,  whitish  layer  forms 
on  the  surface.  When  this  is  well  developed  stains  are  prepared 
from  it,  and  if  the  culture  is  found  to  be  pure  and  uncontaminated 
it  is  evaporated  on  a  water  bath  at  a  temperature  of  70°  to  80°  C. 
down  to  one-tenth  of  its  original  bulk.  The  thick  brown  liquid  so 
obtained,  containing  from  30  to  50  per  cent,  glycerin,  is  first  filtered 
through  chemical  filter  paper  and  then  through  a  Chamberland  or 
Pasteur  filter.  The  product  of  these  manipulations  is  known  as 


FEDERAL  GOVERNMENT  INSPECTION  353 

crude  tuberculin;  it  is  a  fairly  stabile  product,  and  can  be  kept  in  a 
refrigerator  for  a  long  time.  Before  use  it  is  diluted  with  nine  times 
its  bulk  of  a  sterile  J  per  cent,  watery  carbolic-acid  solution.  This  old 
tuberculin  is  today  still  considered  the  best  preparation  for  diagnostic 
use  in  man  and  cattle. 

A  good  tuberculin  should  kill  in  a  minimal  dose  of  0.5  c.c.  a  guinea- 
pig  which  has  been  infected  with  tuberculosis  three  to  four  weeks 
before  the  tuberculin  is  tested.  Death  should  occur  within  six  to 
thirty  hours.  Sometimes  tuberculins  are  much  stronger  and  kill  a 
guinea-pig  previously  infected  (three  to  four  weeks)  in  doses  of  0.1  c.c. 
to  0.05  c.c.  Guinea-pigs  infected  eight  to  ten  weeks  previously  will 
be  killed  in  centigram  doses  of  a  good  strong  tuberculin. 

In  testing  cattle  with  tuberculin  for  latent  tuberculosis  the  following 
doses  of  the  dilute  tuberculin  are  used: 

For  large  bulls  or  steers,  4  c  c 

For  large  cows,  3.5  c.c. 

For  medium-sized  cows  and  steers,  3  c.c. 

For  heifers  and  young  bulls,  one  to  two  years  old,  2  c.c. 

For  calves  under  one  year,  1  c.c. 

For  sheep  and  goats,  0.5  to  1  c.c. 

For  pigs  of  various  ages,  1  to  3  c.c. 

For  pigs  under  four  months,  1  c.c. 

For  pigs  from  four  to  nine  months,  1.5  to  2  c.c. 

For  pigs  from  nine  to  sixteen  months,  2.5  c.c 

For  pigs  above  eighteen  months,  3  c.c. 

Different  brands  of  tuberculin  furnished  by  government  and  private 
institutions  generally  state  the  exact  doses  for  various  animals  at 
various  ages. 

Federal  Government  Directions  for  Inspecting  Herds  with  the  Tuber- 
culin Test. — Inspections  should  be  carried  on  while  the  herd  is  stabled. 
If  it  is  necessary  to  stable  animals  under  unusual  conditions  or  among 
surroundings  that  make  them  uneasy  and  excited,  the  tuberculin 
test  should  be  postponed  until  the  cattle  have  become  accustomed 
to  the  new  conditions.  The  inspection  should  begin  with  careful 
physical  examination  of  each  animal.  This  is  essential,  because  in 
some  severe  cases  of  tuberculosis  no  reaction  follows  the  injection 
of  tuberculin  on  account  of  saturation  with  toxins,  but  experience 
has  shown  that  these  cases  can  be  discovered  by  physical  exami- 
nation. The  latter  should  include  a  careful  examination  of  the 
udder  and  of  the  superficial  lymphatic  glands  and  auscultation  of 
the  lungs. 

Each  animal  should  be  numbered  or  described  in  such  a  way  that 
it  can  be  recognized  without  difficulty.  It  is  well  to  number  the 
stalls  with  chalk  and  transfer  these  numbers  to  the  transfer  sheet, 
so  that  the  temperature  of  each  animal  can  be  recorded  in  its  appro- 
priate place  without  danger  of  confusion.  The  following  procedure 
has  been  used  extensively  and  has  given  excellent  results : 
23 


354  THE  BACILLUS  OF  TUBERCULOSIS 

(a)  Take  the  temperature  of  each  animal  to  be  tested  at  least 
twice  at  intervals  of  three  hours  before  tuberculin  is  injected. 

(6)  Inject  the  tuberculin  in  the  evening,  preferably  between  the 
hours  of  6  and  9  P.M.  The  injection  should  be  made  with  carefully 
sterilized  hypodermic  syringes.  The  most  convenient  point  for  inject- 
ing is  back  of  the  left  scapula.  Prior  to  the  injection  the  skin  should 
be  washed  carefully  with  a  5  per  cent,  solution  of  carbolic  acid  or 
other  antiseptic. 

(c)  The  temperature  should  be  taken  nine  hours  after  the  injection, 
and  temperature  measurements  repeated  at  regular  intervals  of  two 
to  three  hours  until  the  sixteenth  hour  after  the  injection. 

(d)  When  there  is  no  elevation  of  temperature  at  this  time  (sixteen 
hours  after  injection)  the  examination  may  be  discontinued,  but  if 
the  temperature  shows  an  upward  tendency,  measurements  must  be 
continued  until  a  distinct  reaction  is  recognized  or  until  the  tem- 
perature begins  to  fall. 

(e)  If   the  reaction  is  detected  prior  to  the   sixteenth  hour  the 
measurements  should   be   continued    until   the    expiration  of    this 
period. 

(/)  If  there  is  an  unusual  change  of  temperature  of  the  stable, 
or  a  sudden  change  in  the  weather,  this  fact  should  be  recorded  on 
the  report  blank. 

(g)  If  a  cow  is  in  a  febrile  condition  tuberculin  should  not  be 
used,  because  it  would  be  impossible  to  determine  whether,  if  a  rise 
in  temperature  occurred,  it  was  due  to  the  tuberculin  or  to  some 
transitory  illness. 

(Ji)  Cows  should  not  be  tested  within  a  few  days  before  or  after 
calving,  for  experience  has  shown  that  the  result  at  this  time  may 
be'misleading. 

(i)  The  tuberculin  test  is  not  recommended  for  calves  under  three 
months  old. 

(j)  In  old,  emaciated  animals  and  in  re-tests  use  twice  the  usual 
dose,  for  these  animals  are  less  sensitive. 

(&)  Condemned  cattle  must  be  removed  from  the  herd  and  kept 
away  from  those  that  are  healthy. 

(/)  In  making  postmortems  the  carcasses  should  be  thoroughly 
inspected  and  all  the  organs  should  be  examined. 

Reaction  after  Tuberculin. — The  characteristic  reaction  which  occurs 
when  an  animal  affected  with  tuberculosis  receives  an  injection  of 
good  active  tuberculin  in  the  proper  dose  (see  above)  is  the  following : 

The  temperature  begins  to  rise  gradually  six  to  twelve  hours 
after  the  injection;  twelve  to  twenty-one  hours  after  the  injection  it 
reaches  its  maximum;  between  the  twenty-fourth  and  fortieth  hours 
it  returns  to  normal.  There  is,  however,  during  this  interval  between 
the  twenty-fourth  and  fortieth  hour  not  infrequently  a  second  but 
more  moderate  rise.  In  rare  cases  the  first  rise  may  begin  before 
the  sixth  or  after  the  twelfth  hour,  but  these  are  rather  exceptional 


REACTION  AFTER  TUBERCULIN  355 

cases.  The  respiration  and  pulse  often  become  increased  in  relation 
to  the  rise  in  temperature,  at  other  times  these  physiologic  functions 
show  no  marked  changes  from  the  normal,  Sometimes  about  the 
sixth  to  the  eighth  hour  after  the  injection  the  animal  suffers  from 
some  weakness  and  lack  of  appetite.  Trembling  of  the  muscles  may 
also  be  observed.  The  secretion  of  milk  during  a  positive  reaction 
becomes  reduced,  generally  between  3  and  8  per  cent.,  exceptionally 
as  high  as  13  per  cent.  There  is  no  direct  relation  between  the 
intensity  of  the  reaction  and  the  intensity  of  the  tubercular  process, 
except  that  much  advanced  cases  of  tuberculosis  in  weakened  animals 
give  a  weak  reaction. 

The  tuberculin  test  has,  as  a  rule,  no  lasting  ill  effect,  except  in 
very  weak  animals  with  advanced  tuberculosis,  where  the  test  may 
be  followed  by  a  more  or  less  permanent  rise  in  temperature  and  a 
more  rapid  course  of  the  tubercular  process.  Healthy  animals  do 
not  react  or  only  very  slightly  to  the  ordinary  diagnostic  doses  or  to 
even  larger  doses  of  tuberculin;  this  is  also  true  of  animals  suffering 
from  other  diseases.1  It  should  also  be  noted  that  a  positive  reaction 
can  only  be  expected  after  the  tubercular  infection  has  existed  for  a 
certain  minimum  time.  Nocard  showed  by  experiments  that  at  least 
two  weeks  must  elapse  between  the  time  of  infection  with  moderate 
doses  of  tubercle  bacilli  and  the  time  of  the  first  appearance  of  a 
positive  tuberculin  test.  McFadyean  found  a  positive  reaction  in 
eight  days  after  very  large  doses  of  infecting  bacilli.  After  natural 
infections  it  requires  doubtless  a  much  longer  time  before  a  positive 
test  can  be  obtained.  The  limits  given  above  all  refer  to  infection 
by  injection.  According  to  Nocard  and  Rosignol,  infection  through 
the  intestinal  tract  is  followed  by  a  positive  tuberculin  reaction  after 
thirty-two  to  forty-eight  days;  while  infection  by  inhalation  leads  to 
positive  reactions  after  nineteen  to  thirty-two  days. 

Nocard  regards  a  rise  of  1.4°  F.  as  insignificant;  one  from  1.4°  to 
2.5°  F.  (always  provided  that  it  occurred  in  the  typicaj  gradual 
manner)  as  suspicious  and  requiring  re-testing  at  the  end  of  a  month; 
while  a  rise  of  2.5°  to  5.4°  F.  absolutely  indicates  the  presence  of 
a  tubercular  process  in  the  animal  tested. 

The  Eighth  International  Veterinary  Congress  (1905,  Budapest), 
adopted  the  following  resolutions  in  regard  to  the  tuberculin  test: 

1.  The  preparation  and  supply  of  tuberculin  should  be  controlled 
by  the  State. 

2.  No  animal  whose  temperature  exceeds  39.5°  C.  (103°  F.)  is  a 
fit  subject  for  the  tuberculin  test. 

3.  A  rise  of  temperature  to  above  40°  C.  (104°  F.)  in  any  animal 
whose  temperature  at  the  moment  of  injection  was  below  39.5°  C. 
(103°  F.)  is  to  be  regarded  as  a  positive  reaction. 

1  The  tuberculin  test  is,  of  course,  not  applicable  in  the  presence  of  high  fever  no  matter  to 
what  it  may  be  due. 


356  THE  BACILLUS  OF  TUBERCULOSIS 

4.  Any  rise  in  temperature  between  39.5°  C.  (103°  F.)  and  40°  C. 
(104°  F.)  must  be  regarded  as  of  doubtful  significance;  animals 
exhibiting  such  require  special  study. 

The  question  of  how  soon  an  animal  reacting  positively  will  again 
react  has  been  studied  experimentally  by  a  number  of  investigators. 
According  to  Nocard's  experiments  on  24  cows  reacting  positively 
upon  the  first  test,  33  per  cent,  reacted  positively  to  another  test  made 
twenty-four  to  forty- eight  hours  after  the  termination  of  the  first 
test;  50  per  cent,  positively  after  eight  days;  60  per  cent,  positively 
after  two  weeks,  and  100  per  cent,  after  one  month.  According  to 
the  experience  in  the  Prussian  Sea  Quarantine  Station,  100  per  cent, 
react  positively  in  a  second  test  made  almost  immediately  after  the 
first  has  completely  disappeared,  but  five  times  the  amount  of  the 
first  test  dose  must  be  used.  According  to  Valle  the  double  dose  of 
the  first  test  dose  will  give  a  positive  result  when  used  thirty-six  to 
forty-eight  hours  after  the  termination  of  the  first  reaction.  The 
maximum  temperature,  however,  is  reached  much  earlier  in  the 
second  test  and  the  fall  in  temperature  is  more  rapid  than  in  the 
first  test. 

The  accuracy  of  the  conclusions  drawn  from  the  test  varies  accord- 
ing to  the  good  judgment  of  those  who  make  the  test.  According  to 
Bang,  under  satisfactory  conditions  the  positive  diagnosis  upon  the 
basis  of  the  test  is  found  correct  in  96  per  cent.  According  to  Jensen's 
statistics  90.8  per  cent,  of  tubercular  animals  out  of  a  total  of  468 
gave  a  positive  result.  Ever's  statistics  of  563  cases  later  examined 
by  postmortem  examinations  showed  a  correct  interpretation  of  a 
positive  test  in  86.9  per  cent.  The  recent  experience  in  the  United 
States  with  the  tuberculin  test  of  cattle  has  been  even  more  favorable 
than  the  earlier  observations  in  Europe.  Melvin  reported  that  of 
24,784  head  of  cattle  tuberculin-tested  in  1907-08  with  a  positive 
reaction,  24,387,  or  98.39  per  cent.,  upon  being  slaughtered  were 
found  to  be  tubercular.  Nocard  and  Hutyra  and  Marek  state  that 
animals  affected  with  echinococcus  or  actinomycosis  never  react 
positively  to  the  tuberculin  test. 

Ophthalmo-tuberculin  Reaction. — Wolf-Eisner  and  Calmette  almost 
simultaneously  and  independently  introduced  another  form  of 
tuberculin  reaction  into  medical  practice.  It  consists  in  the  instil- 
lation of  one  or  two  drops  of  a  tuberculin  solution  prepared  in  a 
special  manner  from  the  watery  solution  of  an  alcoholic  precipitate  of 
a  killed  tubercle  bacilli  culture  into  the  conjunctival  sac  near  the 
inner  canthus  of  the  eye.  The  test  has  been  found  quite  accurate 
in  man,  and  several  observers  have  tried  it  on  cattle.  Bailliart, 
reporting  to  the  Sixth  International  Tuberculosis  Congress  in  Wash- 
ington, stated  with  reference  to  the  ophthalmo-tuberculin  test  in 
cattle:  "The  ophthalmo-reaction  is  a  diagnostic  procedure  which 
is  usually  without  danger  if  it  is  applied  only  to  eyes  free  from 
tubercular  lesions  of  any  kind.  It  is  sometimes  followed  by  mild  and 


BOVOVACCINE  357 

transitory  accidents.  Very  often  the  reaction  is  doubtful.  In  cattle, 
doubtful  cases,  owing  to  the  difficulty  of  examination,  must  be 
regarded  as  negative.  In  these  animals  a  simple  (primary)  ophthalmo- 
reaction  is  a  very  unreliable  procedure  and  cannot  take  the  place  of 
inoculation  with  tuberculin.  A  secondary  ophthalmo-reaction  gives 
very  much  more  trustworthy  results/'  White  and  McCampbell, 
reporting  on  the  same  subject  to  the  same  Congress,  however,  place 
more  confidence  in  the  primary  test,  but  they  likewise  do  not  consider 
it  of  much  value  in  the  diagnosis  of  tuberculosis  in  cattle.  In  some 
cases  the  reaction  is  very  slight,  in  others  pronounced  congestion 
with  profuse  exudates  is  noted.  They  were  inclined  to  rely  primarily 
on  the  result  of  the  first  instillation  of  tuberculin.  In  the  majority 
of  animals  tested  the  reaction  increases  in  its  intensity  with  each 
subsequent  instillation  of  tuberculin.  This  fact  indicates  the  develop- 
ment of  a  local  hypersusceptibility,  or  anaphylaxis,  associated  with  a 
partial  immunity. 

Pirquet's  Cutaneous  Tuberculin  Test. — This  consists  in  the  vaccination 
with  a  few  drops  of  tuberculin  on  the  arm,  and  has  been  extensively 
used  in  children  but  very  little  in  animals.  The  same  is  true  of  the 
so-called  dermo-reaction,  where  a  tuberculin-lanolin  ointment  is  rubbed 
into  the  skin.  These  two  methods,  like  the  ophthalmo-tuberculin 
test,  are  of  no  great  value  in  the  detection  of  latent  tuberculosis  in 
domestic  animals. 

IMMUNIZATION  AND  PROTECTIVE    INOCULATION. 

Bovovaccine. — Starting  from  the  viewpoint  that  the  bacillus  of 
human  tuberculosis,  derived  from  the  sputum  of  tubercular  patients, 
is  not  very  virulent  for  cattle,  von  Behring  has  worked  out  a  method 
of  protective  and  immunizing  inoculation.  Since  tubercle  bacilli 
of  human  derivation,  even  apart  from  those  comparatively  not 
numerous  cases  of  infection  by  bacilli  of  the  bovine  type,  vary  a  good 
deal  in  their  effect  upon  cattle,  it  is  not  a  matter  of  indifference 
what  stem  of  human  tubercle  bacilli  is  selected  for  the  preparation 
of  a  vaccine  to  be  used  on  bovines.  Von  Behring,  after  many  pre- 
liminary experiments,  selected  a  culture  of  a  low  degree  of  virulency 
for  cattle  which  had  never  led  to  any  untoward  accidents.  From 
transplants  of  this  original  and  extensively  tested  culture  all  his 
bovovaccine  has  been  prepared.  The  experience  of  von  Behring  and 
his  co-workers  and  of  those  who  have  tried  his  method  and  vaccine, 
like  Hutyra,  a  French  commission  and  others,  has  been  very  favorable 
both  as  to  the  harmlessness  of  the  procedure  and  as  to  the  protection 
which  it  affords  against  natural  infection  or  artificial  inoculation 
with  the  bovine  tubercle  bacillus.  The  bovovaccine  is  prepared  from 
a  four  to  six-weeks-old  glycerin  bouillon  culture  of  the  selected  human 
tubercle  bacillus.  The  culture  is  filtered  and  the  bacilli  remaining 
on  the  filter  are  dried  in  vacuo  over  sulphuric  acid  for  twenty-four 


358  THE  BACILLUS  OF  TUBERCULOSIS 

hours.  The  mixture  is  then  somewhat  triturated  and  definite  amounts 
are  placed  in  small  glass  tubes.  These  either  contain  five  or  twenty 
immunity  units  (J.  E.).1  One  immunity  unit  (J.  E.)  is  equal  to 
0.004  gram  dry  tubercle  bacilli.  It  is  the  dose  used  for  the  first 
inoculation  of  young  cattle,  while  a  dose  of  0.02  gram  or  5  immunity 
units  is  employed  for  the  second  inoculation,  which  follows  the  first 
after  an  interval  of  twelve  weeks.  The  vaccine  is  prepared  from  the 
dry  bacilli  by  mixing  the  contents  of  the  tubes  with  sterile  physiologic 
salt  solution,  so  that  5  J.  E.  are  mixed  with  10  c.c. ;  20  J.  E.  with  40  c.c. 
salt  solution.  For  the  first  inoculation  2  c.c.  of  the  salt  solution 
bacilli  emulsion  are  used;  for  the  second  inoculation  after  twelve 
weeks  10  c.c.  of  the  emulsion.  Only  healthy  animals  with  a  normal 
temperature  should  be  inoculated.  The  inoculation  is  made  into  the 
jugular  vein.  It  is  hardly  necessary  to  point  out  that  the  mixing 
of  the  dry  bacilli  and  the  injection  of  the  emulsion  must  be  done 
under  all  possible  aseptic  precautions. 

Weber  and  Titze  who  have  employed  the  von  Behring  method  of 
cattle  immunization  believe  that  the  immunity  conferred  by  it  does 
not  last  over  two  years.  Smith  vaccinated  35  calves,  weighing  from 
58  to  284  pounds,  with  seven  different  human  and  three  attenuated 
bovine  cultures,  and  from  his  experimental  work  he  draws  the  follow- 
ing conclusions: 

1.  Vaccination  of  calves  with  the  human  type  of  the  tubercle 
bacillus  is  harmless.    Cases  in  which  injuries  are  said  to  have  resulted 
from  it  may  have  been  due  to  other  concomitant  affections,  among 
which  pneumonia  is  probably  the  most  common.     Persons  trying 
vaccination   should  first  assure   themselves   that   the   culture   they 
intend  to  use  belongs  to  the  human  and  not  to  the  bovine  type  of  the 
bacillus. 

2.  Vaccination  with  the  human  type  of  bacillus  leads  to  a  relatively 
high  resistance  to  fatal  doses  of  the  bovine  bacillus. 

3.  Vaccination  with  carefully  tested  attenuated  bovine  bacilli  may 
be  as  efficacious  even  in  a  single  injection  as  the  double  vaccination 
with  human  bacilli.     Such  vaccination  may  be  less  dangerous  to 
man  than  when  human  bacilli  are  used. 

4.  The  immunity  conferred  by  vaccination,  as  hitherto  practised, 
does  not  appear  to  be  satisfactory  as  regards  degree  or  duration. 
More  evidence  is  needed  with  regard  to  these  points.    The  herds  of 
large  public  institutions  are  well  adapted  to  decide  these  questions 
if  vaccination  is  thoroughly  applied,  and  the  animals  supervised  by 
properly  trained  men. 

5.  Insufficient  immunity  following  vaccination  may  prove  dangerous 
in  giving  rise  to  mild  cases,  after  ordinary  exposure  in  infected  herds, 
which  tend  to  discharge  tubercle  bacilli  from  small  foci  in  the  lungs. 

1  The  term  immunity  unit  (J.  E.)  as  used  in  connection  with  the  bo vo vaccine  of  von  Behring 
differs  in  meaning  from  the  identical  term  as  used  to  express  the  immunizing  value  of  diph- 
theria or  tetanus  antitoxins. 


PROPHYLAXIS  AND  ERADICATION  OF  TUBERCULOSIS     359 

6.  The    immunity    acquired    by    two    vaccinations    with   human 
bacilli  should  be  fortified  by  a  subsequent  injection  of  attenuated 
bovine  bacilli. 

7.  Investigations  should  be  made  looking  toward  the  selection, 
by  the  injection  of  attenuated  bovine  bacilli,  of  races  or  breeds  of 
cattle  which  posses  naturally  a  high  degree  of  resistance  to  tuber- 
culosis.   The  capacity  of  different  breeds  to  acquire  a  high  degree  of 
immunity  should  also  be  investigated. 

8.  The  survival  of  human  and  bovine  bacilli  in  the  lungs  and 
udders  of  calves  vaccinated  intravenously  with  them  should  be  more 
definitely  determined. 

9.  Vaccines  may  be  easily  and  cheaply  prepared  in  the  form  of 
suspensions  in  fluids  ready  for  injection.    The  length  of  time  during 
which  suspensions  maintain  their  highest  efficiency  remains  to  be 
determined. 

Prophylaxis  and  Eradication  of  Tuberculosis  among  Domestic  Animals. 
—The  recognition  of  the  fact  that  tuberculosis  is  an  infectious  disease, 
due  to  a  specific  bacillus  or  to  several  varieties  of  a  single  species, 
has  led  to  effective  measures  to  prevent  the  spread  of  the  disease, 
particularly  after  the  extensive  and  excellent  work  of  Cornet  had 
shown  that  the  tubercle  bacillus  is  not  ubiquitous  but  only  present 
where  it  is  disseminated  by  tubercular  persons  or  animals.  Human 
tuberculosis  has  been  reduced  particularly  by  such  measures  as  the 
destruction  of  the  tubercular  sputum,  the  prevention  of  its  broadcast 
dissemination,  the  isolation  to  a  certain  extent  of  tubercular  persons, 
the  avoidance  of  overcrowding  of  human  residences,  and  the  proper 
use  of  direct  and  diffuse  sunlight  as  a  disinfectant  against  the  tubercle 
bacillus.  Efforts  have  also  been  made  on  a  large  scale  to  prevent 
tuberculosis  and  limit  its  spread  among  the  meat  and  milk-producing 
animals.  These  efforts  are  justified  and  demanded  imperatively,  not 
merely  because  there  is  a  certain  amount  of  danger  of  the  spread  of 
tuberculosis  from  domestic  animals  to  man  (though  the  principal 
danger  among  mankind  is  the  infected  person),  but  also  because 
tuberculosis  among  domestic  animals  is  a  constant  source  of  great 
national  loss,  and  its  eradication  would  be  a  great  purely  economic 
gain  apart  from  all  hygienic  considerations.  An  elaborate  plan  for ' 
finally  stamping  out  tuberculosis  among  cattle  has  been  devised 
by  Bang.  The  method  consists  in  a  careful  clinical  examination  of 
all  cattle  and  subjection  of  the  animals  to  the  tuberculin  test.  Those 
animals  which  already  show  well-marked  clinical  evidence  of  tuber- 
culosis, particularly  of  the  lungs,  intestines,  uterus,  and  udder,  are 
separated  from  the  others  and  slaughtered  as  soon  as  possible.  The 
apparently  healthy  animals  which  do  not  react  to  the  tuberculin  test 
are  likewise  placed  by  themselves  in  a  separate  class.  If  possible 
they  are  kept  in  a  separate  barn,  but  if  this  is  impracticable,  they 
are  at  least  divided  from  the  others  by  a  partition  wall  in  a  space 


360  THE  BACILLUS  OF  TUBERCULOSIS 

with  separate  entrance.  After  the  removal  of  the  evidently  tuberculous 
animals  and  those  which  have  reacted  to  the  test  the  place  where 
the  healthy  animals  are  segregated  should  be  thoroughly  disinfected 
so  that  any  remaining  tubercle  bacilli  may  be  destroyed.  The  more 
thorough  the  segregation,  even  to  the  extent  of  having  their  own 
separate  attendants,  the  better.  Animals  which  have  reacted  to  the 
tuberculin  tests  but  are  otherwise  in  good  condition  may  be  used  for 
breeding  purposes.  The  calves,  however,  should  be  separated  from 
their  tubercular  mothers  within  twenty-four  hours  after  birth.  They 
must  then  be  brought  up  either  on  the  raw  milk  of  healthy  cows  or,  if 
on  their  mother's  milk,  it  must  be  pasteurized  at  a  temperature  which 
will  safely  destroy  the  tubercle  bacilli.  If  any  of  the  calves  show 
diarrhea  they  must  be  separated  immediately  from  the  other  young 
animals.  As  soon  as  possible  the  calves  themselves  are  subjected  to 
the  tuberculin  test  and  the  few  which  react  positively  must  be  separated 
from  the  healthy  ones.  From  time  to  time  the  healthy  cows  must  be 
re-tested  with  tuberculin  so  as  to  forestall  a  clandestine  appearance  and 
spread  of  tuberculosis  among  the  non-infected  stock.  If  this  method 
is  carried  out  persistently,  intelligently,  and  carefully  it  will,  in  a  few 
generations,  very  much  reduce  tuberculosis  in  a  herd  and  will  event- 
ually lead  to  its  ultimate  eradication.  The  results  in  Denmark,  under 
Bang's  personal  supervision,  have  been  very  excellent.  Hutyra  and 
Marek  report  brilliant  results  from  the  Hungarian  State  Cattle  Breeding 
Farm.  In  the  spring  of  1898  the  first  tuberculin  test  of  329  cows 
showed  44.8  per  cent,  infected;  a  later  test  of  647  head  showed  26.6 
per  cent,  infected  animals.  In  the  fall  of  1901,  1042  head  showed 
3.1  per  cent,  infected,  and  the  test  in  1903,  of  1132  animals,  showed 
only  1.8  per  cent,  infected.  In  other  words,  during  five  years  the 
number  of  animals,  without  outside  additions,  had  increased  very 
much,  and  tuberculosis  had  decreased  88  per  cent.  These  are,  indeed, 
encouraging  figures. 

Prevention  of  Tuberculosis  among  Swine. — Among  the  more  important 
precautions  to  prevent  the  spread  of  tuberculosis  among  swine  are 
the  following:  The  animals  should  not  be  allowed  to  feed  after 
tubercular  cattle  nor  to  devour  the  carcasses  of  cows  dead  from 
tuberculosis,  and  skimmed  milk  from  non-tested  cows  should  not  be 
fed  to  hogs  until  it  has  been  properly  pasteurized  or  sterilized. 

A  community  or  State  which  does  not  try  to  prevent  the  spread  of 
tuberculosis  and  make  proper  attempts  to  stamp  it  out  ultimately 
among  its  food  and  milk-producing  domestic  animals  by  proper 
equitable  regulations,  ordinances,  and  statutory  laws  is  sorely  lacking 
in  doing  its  duty  toward  its  citizens,  the  more  so  because  there  are 
States  where  such  laws  are  in  existence.  This  must  necessarily 
operate  in  such  a  manner  that  the  non-protected  territory  will  become 
the  dumping  ground  for  tubercular  animals  condemned  elsewhere. 


BOVINE  TUBERCULOSIS  361 


THE    IDENTITY    OR   NON-IDENTITY    AND    INTERTRANSMISSI- 
BILITY  OF  TUBERCLE  BACILLI  FROM  VARIOUS  SOURCES. 

Bovine  Tuberculosis. — When  Robert  Koch,  in  1882,  made  public 
his  work  in  connection  with  the  discovery  of  the  tubercle  bacillus  he 
believed  that  tuberculosis  of  man  and  animals  was  an  absolutely 
identical  disease  and  that  tubercle  bacilli,  no  matter  from  what 
source  they  were  derived,  were  virtually  identical  in  each  and  every 
respect.  Smith,  in  1896,  was  the  first  to  call  attention  to  the  fact 
that  tubercle  bacilli  from  human  tubercular  sputum  and  those 
derived  from  bovine  tubercular  lesions  present  certain  differences  in 
their  morphology,  biology,  and  virulency  toward  various  animals.  A 
more  extensive  communication  concerning  this  work  followed  in  1898, 
and  another  report  was  published  in  1905.  Smith's  work  as  to  differ- 
ences in  virulency  toward  domestic  animals  has  since  been  confirmed 
by  Dinwiddie  and  others,  and  it  has  been  established  that  tubercle 
bacilli  from  bovine  sources  are  generally  short,  straight,  and  cylindrical 
in  outline.  At  first  they  grow  rather  poorly  on  artificial  media,  are  less 
influenced  by  variations  in  the  composition  of  the  medium,  and  tend 
to  remain  short  when  grown  on  artificial  media  for  a  number  of  genera- 
tions. Bacilli  derived  from  human  tubercular  sputum,  on  the  other 
hand,  are  generally  more  slender  from  the  beginning,  often  curved. 
They  grow  much  more  abundantly  in  first  generations  on  artificial 
media  and  show  a  tendency  to  remain  slender  or  to  become  so  if  they 
did  not  show  this  form  in  a  decided  manner  from  the  beginning. 
Mohler  and  Washburn,  in  a  comparative  study  of  tubercle  bacilli  from 
varied  sources,  have  come  to  the  following  conclusions  as  to  a  certain 
plastic  variability  in  their  morphologic  features,  and  say : 

"While  certain  peculiarities  of  growth,  morphology,  and  patho- 
genesis  are  observed  with  a  fair  degree  of  constancy  in  bacilli  of 
human  origin,  nevertheless  these  characteristics  are  not  universal, 
and  notable  exceptions  are  observed  which  would  confuse  those  who 
would  attempt  to  establish  their  origin  by  means  of  such  character- 
istics. A  similar  degree  of  constancy  in  the  morphologic,  biologic, 
and  pathogenic  characters  of  the  bovine  bacillus  is  generally  noted; 
but  a  certain  range  of  differences  has  been  observed,  which,  though 
apparently  more  limited  than  for  the  human  bacillus,  is  nevertheless 
suggestive  of  aberrant  forms." 

An  important  biologic  difference  in  the  action  of  human  and  bovine 
tubercle  bacilli  in  changing  the  reaction  of  the  culture  medium  is 
described  by  Smith  as  follows :  "When  flasks  of  glycerin  bouillon  in 
layers  1 J  to  2J  centimeters  deep  are  inoculated  with  scales  of  tubercle 
bacilli  from  glycerin-agar  cultures  the  floating  masses  soon  begin  to 
expand,  and  after  one  or  two  months  a  complete  membrane  will  have 
formed  over  the  relatively  clear  bouillon.  The  bacillar  masses  which 
fall  to  the  bottom  at  the  outset  increase  but  slightly  in  size."  This 


362  THE  BACILLUS  OF  TUBERCULOSIS 

mode  of  surface  growth  of  tubercle  bacilli  was  shared  by  all  mam- 
malian races  examined  by  Smith  after  they  had  acquired  a  certain 
saprophytic  habit,  and  the  process  is  well  known  to  all  who  have 
cultivated  tubercle  bacilli  for  the  preparation  of  tuberculin.  If 
ordinary  bouillon  prepared  from  fresh  beef  to  which  3  to  5  per  cent, 
glycerin  has  been  added  is  used,  and  if  the  acidity  is  made  equiva- 
lent to  about  2  per  cent,  of  normal  acid,  phenolphthalein  being  the 
indicator,  the  reaction  of  the  bouillon  during  the  formation  of  the 
membrane  will  take  one  of  two  directions.  It  may  approach  the 
neutral  point  and  reach  an  acidity  or  an  alkalinity  of  about  0.1  to 
0.2  per  cent,  when  the  membrane  is  complete  and  remain  at  this 
point,  or  else  the  acidity  may  diminish  during  the  first  months, 
increase  again  during  the  second,  and  fluctuate  more  or  less  if  the 
observation  is  continued,  but  the  reaction  does  not  reach  the  neutral 
point.  In  the  human  cultures,  on  the  other  hand,  the  reaction  curve 
also  moves  toward  the  neutral  point,  but  soon  swings  back  to  a  greater 
acidity. 

The  human  type  of  tubercle  bacillus  has  very  slight  virulency 
toward  cattle.  This  fact  may  indeed  be  considered  as  established. 
It  has  been  proved  beyond  question  that  human  sputum  bacilli  used 
on  cattle  in  doses  in  which  bovine  bacilli  will  produce  general  tuber- 
culosis have,  as  an  almost  universal  rule,  practically  no  lasting  effects 
whatever.  Bovine  tubercle  bacilli  are  likewise  more  virulent  than 
the  human  type  toward  guinea-pigs,  rabbits,  mice,  rats,  sheep,  goats, 
pigs,  dogs,  equines,  etc.  Rabbits  are  particularly  well  adapted  to 
show  the  difference  in  virulency  between  the  bovine  and  human 
types  when  applied  to  the  same  species  of  animals.  According  to 
Weber,  0.001  gr.  of  bovine  bacilli  injected  intravenously  kills  rabbits 
with  the  production  of  a  general  tuberculosis  in  three  weeks,  while 
the  human  type  under  the  same  conditions  produces  a  very  chronic 
slow  form  of  tuberculosis  only  after  months.  The  first  extensive 
experiments  showing  that  such  is  the  action  of  human  sputum 
bacilli  toward  cattle  were  made  by  Koch  and  Schiitz.  Upon  the 
basis  of  these  observations  Koch,  in  1901,  before  the  British  Congress 
on  Tuberculosis  appears  to  have  taken  the  standpoint  that  the 
danger  of  transmitting  bovine  tuberculosis  to  man  is  practically  nil. 
After  1901,  however,  it  was  ascertained,  and  Ravenel  had,  indeed, 
made  some  observations  before  this  time,  that  tubercle  bacilli 
of  the  bovine  type,  very  virulent1  to  cattle,  are  found  in  certain 
tubercular  lesions  in  man.  Such  bacilli,  however,  with  a  possible 
single  exception  (Arloing),  have  never  been  found  in  the  sputum  of 
tubercular  patients,  and  since  human  sputum  bacilli  were  exclusively 
used  in  the  early  experiments  of  Koch  and  Schiitz  the  former  was 
induced  to  draw  conclusions  which  were  too  radical  and  which 
cannot  be  upheld  at  the  present  time.  Koch,  in  1908,  at  the  Inter- 

1  Ravenel  found  such  bacilli  in  the  mesenteric  glands  of  a  child. 


BOVINE  TUBERCULOSIS  363 

national  Congress  at  Washington,  had  evidently  modified  his  stand- 
point, which  he  then  expressed  as  follows : 

"1.  The  tubercle  bacilli  of  bovine  tuberculosis  are  different  from 
those  of  human  tuberculosis.  2.  Human  beings  may  be  infected  by 
bovine  tubercle  bacilli,  but  serious  diseases  from  this  cause  occur 
very  rarely.  3.  Preventive  measures  against  tuberculosis  should 
therefore  be  directed  primarily  against  the  propagation  of  human 
tubercle  bacilli." 

Since  1901  a  great  number  of  experiments  have  been  made  in 
relation  to  the  question  of  the  difference  in  virulency  between  human 
and  bovine  tubercle  bacilli,  the  possibility  of  infecting  cattle  with 
bacilli  of  human  derivation,  and  presence  or  absence  of  bacilli  of 
the  bovine  type  in  human  tubercular  lesions.  Those  who  followed 
Koch  and  Schiitz  did  not  confine  themselves  to  human  sputum 
bacilli,  but  also  chose  tubercular  material  from  other  sources,  and  it 
was  soon  found  that  such  material  sometimes  contained  bacilli  of  the 
bovine  type  which  are  quite  virulent  to  cattle.  Kossel,  Weber  and 
Heuss,  after  Koch's  and  Schiitz '  experiments,  examined  56  cases  of 
human  tuberculosis  and  found  in  49  bacilli  of  the  human  type,  in 
5  bacilli  of  the  bovine  type,  and  in  2  bacilli  of  both  types.  The  5 
cases  in  which  they  found  bacilli  of  the  bovine  type  were  those  of 
children  under  five  years  of  age,  4  of  which  warranted  the  conclusion 
that  the  bacilli  had  gained  entrance  through  the  intestinal  tract. 
The  49  cases  in  which  bacilli  of  the  human  type  only  had  been  found 
included  many  forms  of  tuberculosis,  such  as  tuberculosis  of  the 
lungs,  lymph  glands,  bones,  joints,  genito-urinary  tract,  intestinal 
tract,  peritoneum,  and  meninges,  and  general  miliary  tuberculosis. 
The  tubercular  subjects  were  of  all  ages,  and  among  them  2  cases  of 
tuberculosis  of  the  peritoneum  were  found  which  anatomically  were 
of  the  "Perlsucht"  type,  but  in  which  the  bacilli  were  not  of  the  bovine 
type  but  the  human  kind. 

Weber,  in  the  article  on  "Tuberculosis  of  Man  and  Animals," 
in  Kolle  and  Wassermann's  Manual  of  Bacteriology,  enumerates, 
including  his  own,  14  cases  of  tuberculosis  in  man,  collected  from 
literature,  in  which  bacilli  of  the  bovine  type  had  been  isolated  beyond 
doubt.  Since  the  publication  of  his  report  several  further  cases  have 
been  added  to  the  list  by  Smith  and  Mohler  and  Washburn,  and 
perhaps  by  some  others,  but  it  is  safe  to  say  that  there  are  not  many 
above  50  cases  now  on  record,  and  none  of  these  were  cases  of  pul- 
monary human  tuberculosis.  It  appeared  to  be  the  general  consensus 
of  opinion  at  the  last  International  Congress  on  Tuberculosis  that 
Koch's  contention  was  correct  and  that  no  case  of  human  pulmonary 
tuberculosis  had  yet  been  traced  beyond  doubt  to  the  bovine  tubercle 
bacilli,  because  the  few  cases  reported  as  such  were  not  beyond 
reasonable  criticism.  The  cases  of  human  tuberculosis  shown  to  be 
caused  by  bacilli  of  the  bovine  type  are,  as  appears,  all  traceable  to 
the  entrance  of  the  bovine  bacilli  through  the  intestinal  tract,  probably 


364  THE  BACILLUS  OF  TUBERCULOSIS 

with  highly  contaminated  milk.  It  must,  therefore,  be  considered 
as  established  that  man  can  be  and  is  occasionally  infected  with 
bovine  tuberculosis  and  that  tubercular  cattle,  particularly  cows,  are 
not  a  negligible  source  of  danger  to  man  but  one  which  warrants 
measures  to  prevent  the  spread  of  tuberculosis  from  cattle  to  man. 

Avian  Tuberculosis. — There  are  certain  differences  in  the  morphology 
and  biology  of  the  avian  tubercle  bacillus  compared  with  the  mam- 
malian races.  Microscopically  it  resembles,  on  the  whole,  the  human 
type  rather  than  the  bovine,  but  very  often  it  shows  considerable  pleo- 
morphism,  with  numerous  coarse,  club-shaped,  and  branched  forms. 
It  grows  more  rapidly  in  artificial  cultures  than  either  the  human  or 
the  bovine  type,  and  develops,  in  ten  to  fifteen  days,  round,  moist 
colonies,  which  grow  rapidly  and  then  form  a  continuous,  dull-shining, 
wax-like,  fatty  film  over  the  surface  of  the  medium,  which  later 
becomes  corrugated  and  assumes  a  decidedly  yellow  color.  In  its 
moist  character  cultures  of  avian  tubercle  bacilli  vary  greatly  from 
the  dry  cultures  of  mammalian  organisms.  The  latter  do  not  grow 
at  temperatures  over  41°  C.,  while  the  former  still  multiply  at  temper- 
atures between  45°  and  50°  C.  Guinea-pigs  are  quite  resistant 
toward  avian  tubercle  bacilli,  but  they  sometimes  contract  the  avian 
disease  in  artificial  inoculation  and  die  from  it.  Rabbits  appear  to  be 
more  susceptible  to  avian  than  to  human  tubercle  bacilli.  The  horse, 
according  to  Nocard,  is  susceptible  to  avian  tubercle  bacilli  in  artificial 
infection.  Chickens  and  pigeons  are  very  susceptible  and  develop 
the  typical  picture  of  avain  tuberculosis.  These  birds  are  generally 
not  very  susceptible  to  bacilli  of  human  derivation,  though  some 
observers  have  had  positive  results  in  inoculation  experiments,  and 
it  is  also  claimed  that  barnyard  fowl  have  been  infected  from  the 
sputum  of  tubercular  patients.  Straus  and  Wurz  and  Nocard,  how- 
ever, have  fed  chickens  for  a  long  time  on  tubercular  sputum  without 
producing  the  disease.  Nocard  implanted  collodion  sacs  containing 
human  tubercle  bacilli  in  the  peritoneal  cavity  of  chickens,  and 
succeeded  in  changing  their  morphologic  and  cultural  characteristics 
and  their  pathogenicity  for  chickens  so  that  they  became  much  like 
the  bacilli  of  avian  tuberculosis.  Mohler  and  Washburn  investigated 
an  outbreak  of  tuberculosis  among  the  fowl  of  a  large  ranch  in 
Oregon.  This  epizootic  seemed  to  extend  to  the  swine  of  the  same 
place,  through  feeding  the  hogs  on  the  carcasses  of  fowl  that  suc- 
cumbed to  the  disease.  They  found  that  young  pigs  could  be  infected 
with  tuberculosis  by  being  fed  material  from  tuberculous  chickens 
from  this  ranch,  and  that  these  avian  bacilli,  after  a  repeated  passage 
through  mammals,  would  produce  typical  lesions  of  tuberculosis  in 
the  latter. 

From  the  various  observations  quoted  above  it  appears  that  avian 
tuberculosis  can  be  transmitted  to  mammals,  and  vice  versa.  It  is, 
of  course,  an  established  fact  that  parrots  in  captivity  are  quite 
susceptible  to  tubercular  infections  from  man. 


QUESTIONS  365 


QUESTIONS. 

1.  Describe  the  morphology  of  the  tubercle  bacillus? 

2.  Why  have  some  authors  classified  the  tubercle  bacillus  under  streptothricse  ? 

3.  Describe  spore  formation  of  the  tubercle  bacillus  and  its  flagella. 

4.  Why  is  the  tubercle  bacillus  difficult  to  stain? 

5.  What  is  an  acid -fast  bacterium? 

6.  What  reagents  are  needed  for  staining  the  tubercle  bacillus? 

7.  Describe  the  steps  in  staining  the  bacillus  in  sputum,  pus,  caseous  material, 
etc. 

8.  Describe   Biedert's  liquefaction  and   sedimentation  method  for  finding 
a  few  tubercle  bacilli. 

9.  Describe  the  method  of  staining  tubercle  bacilli  (a)  in  paraffin  sections; 
(6)  in  celloidin  sections. 

10.  Describe  the  method  of  obtaining  a  first  generation  of  a  pure  culture  of 
tubercle  bacilli  from  tubercular  pathologic  material. 

11.  What  are  the  media  used  for  obtaining  the  bacillus  in  pure  cultures? 

12.  Upon  what  do  the  toxic  effects  of  the  tubercle  bacillus  depend? 

13.  Discuss  the  resistance  of  the  tubercle  bacillus. 

-  14.  What  disinfectants  are  efficient  in  the  destruction  of  the  tubercle  bacillus? 
Which  are  inefficient? 

15.  Describe  the  preparation  of  Koch's  old  tuberculin  used  in  making  the 
tuberculin  test  in  cattle. 

16.  What  are  the  doses  to  be  used  on  animals  of  various  ages  and  sizes? 

17.  What  is  the  standard  of  a  good  reliable  tuberculin? 

18.  Describe  the  steps  in  making  the  tuberculin  test  in  cattle. 

19.  Discuss  the  interpretation  of  the  result  of  the  test. 

20.  How  soon   will   an   animal  react   positively  again   after  once   reacting 
positively? 

21.  WTiat  is  the  accuracy  of  the  tuberculin  test  in  "cattle? 

22.  What  is  the  ophthalmo-tuberculin  reaction?    What  is  its  value  in  cattle? 

23.  What  is  the  von  Pirquet  test? 

24.  Discuss  the  preparation,  use,  and  value  of  von  Behring's  bovovaccine. 

25.  What  is  the  opinion  of  various  investigators  concerning  this  method  of 
protective  inoculation  ? 

26.  What  has  been  the  most  successful  single  measure  in  limiting  the  spread 
of  tuberculosis  (pulmonary)  in  man  ? 

27.  Describe  in  detail  the  method  of  Bang  for  limiting  the  spread  of  tuber- 
culosis among  cattle  and  finally  stamping  it  out  entirely. 

28.  What  measures  should  be  taken  to  prevent  the  spread  of  tuberculosis 
among  swine? 

29.  What  are  the  differences  in  morphology  between  the  so-called  human 
type  and  the  so-called  bovine  type  of  tubercle  bacilli? 

30.  What  are  their  cultural  differences? 

31.  What  is  their  difference  as  to  acid  production  in  glycerin  bouillon? 

32.  What  is  the  virulency  of  the  human  tubercle  bacilli  toward  cattle? 

33.  What  is  the  comparative  virulency  of  the  human  and  the  bovine  type 
toward  rabbits? 

34.  What  toward  pigs,  guinea-pigs,  and  equines? 

35.  What  was  the  result  of  Koch  and  Schiitz's  inoculation  experiments  with 
human  sputum  bacilli  on  cattle? 

36.  Is  bovine  tuberculosis  ever  transmitted  to  man?    If  so,  in  what  kind  of 
cases  of  human  tuberculosis  has  such  transmission  been  found? 

37.  What  percentage  of  human  pulmonary  tuberculosis  can  be  traced  to  cattle? 

38.  What  was  the  result  of  Kossel,  Weber,  and  Heuss'  researches  and  experi- 
ments with  a  variety  of  human  tubercular  material  as  to  its  human  or  bovine 
origin  ? 

39.  Have  other  cases  of  similar  type  been  reported,  and  by  whom? 

40.  Mention  some  of  the  differences  between  avian  and  mammalian  tubercle 
bacilli,  both  morphologic  and  cultural. 

41.  Are  avian  and  mammalian  tuberculosis  intertransmissible,  and,  if  so,  what 
do  we  know  about  it? 

42.  Describe  Nocard's  method  of  changing  human  tubercle  bacilli  into  the 
avian  type. 


CHAPTEK    XXXI. 

PSEUDOTUBERCULOSIS  AND  ACID-FAST  BACILLI  OTHER  THAN 

THE  TUBERCLE  BACILLUS— RAT  LEPROSY— CHRONIC 

BACTERIAL  DYSENTERY  (JOHNE'S 

DISEASE)  IN  CATTLE. 

Pseudotuberculosis. — When,  after  the  discovery  of  the  tubercle 
bacillus,  numerous  investigators  inoculated  tubercular  material,  or 
what  appeared  to  be  such,  into  laboratory  animals  it  was  ascertained 
that  these  sometimes  developed  lesions  which  on  first  sight  appeared 
to  be  tubercular,  but  which  on  careful  examination  were  found  not  to 
contain  the  tubercle  bacillus  but  other  microorganisms.  Observations 
of  this  kind  multiplied,  and  it  has  become  customary  to  classify 
such  pseudotubercular  pathologic  changes  due  not  to  tubercle  bacilli, 
but  to  entirely  different  organism  as  pseudo tuberculoses.  It  was 
further  found  that  such  pseudotubercular  infections  occurred  not  only 
after  artificial  inoculations,  but  also  spontaneously  as  laboratory 
epizootics  or  among  domestic  and  wild  animals. 

Preisz,  after  having  discovered  a  bacterium  of  this  kind,  and  after 
reviewing  the  work  of  others  in  this  field,  divided  the  bacillary  pseudo- 
tuberculoses  into  the  three  following  groups  based  upon  the  causative 
microorganisms : 

I.  Pseudotuberculoses    due    to    the    Bacillus    pseudotuberculosis 
rodentium  (rodents)  of  Pfeiffer,  also  called  the  Streptobacillus  pseudo- 
tuberculosis  of  Dor. 

II.  Pseudotuberculoses    due    to    the    Bacillus    pseudotuberculosis 
murium  (mice)  of  Kutscher. 

III.  Pseudotuberculoses  due  to  the  Bacillus  pseudotuberculosis  ovis 
(sheep)  of  Preisz. 


BACILLUS  PSEUDOTUBERCULOSIS  RODENTIUM. 

Occurrence. — The  bacillus  causing  pseudotuberculosis  in  rodents 
is  evidently  a  saprophyte  encountered  extensively  in  the  outside 
world.  It  has  been  found  in  garden  earth,  the  sediments  from  rivers, 
contaminated  by  sewage,  dust,  fodder,  milk,  etc.  It  apparently 
becomes  pathogenic  occasionally,  and  then  causes  pseudotubercular 
lesions  in  rabbits,  hares,  cats,  chickens,  pigeons,  and  even  in  cattle, 
swine,  and  other  animals. 

Pathologic  Lesions. — The  pathologic  lesions  which  lead,  as  a  rule, 
to  progressive  emaciation  and  finally  death,  are  preferably  found 


BACILLUS  PSEUDOTUBERCULOSIS  RODENT1UM  367 

in  the  liver  and  spleen.  Here  numerous  whitish  round  nodules  are 
formed,  varying  in  size  from  a  millet  seed  to  a  pea.  They  are  sharply 
defined  from  the  surrounding  tissue,  project  somewhat  above  the 
surface,  and  contain  a  caseous  centre.  Such  nodules  are  also  occasion- 
ally found  in  the  kidneys.  The  abdominal  lymphatic  glands  are 
enlarged,  and  likewise  contain  nodules  which  have  a  tendency  to 
become  confluent.  Nodules  are  also  found  in  the  intestinal  wall, 
and  particularly  in  the  rabbit  the  appendix  is  a  favorable  seat  for 
them.  They  also  involve  the  intestinal  mucosa  where  they  have 
their  seat  in  the  lymph  follicles.  The  lungs  may  likewise  be  affected, 
and  the  process,  according  to  Ligniere,  may  lead  to  purulent  pleuritis 
and  peritonitis.  The  lungs,  according  to  Nocard,  are  particularly 
the  seat  of  numerous  nodules  in  pseudotuberculosis  of  chickens. 

Morphology. — The  Bacillus  pseudotuberculosis  rodentium  of  Pfeiffer 
Is  a  short,  plump  rod  of  small  size,  generally  1  to  2  micra  long,  with 
rounded  ends.  In  older  cultures  and  in  the  tissues  ovoid  forms  are 
seen.  It  has  a  marked  tendency  to  form  shorter  or  longer  chains, 
hence  Dor  called  it  a  streptobacillus.  It  does  not  show  any  marked 
motility  in  the  hanging  drop,  but  Klein  claims  to  have  been  able  to 
demonstrate  one  or  two  flagella  with  the  aid  of  van  Ermengem's 
silver  impregnation  method.  The  organism  can  be  stained  with  the 
ordinary  watery  anilin  dyes;  it  often  shows  polar  or  peripheral  staining, 
particularly  the  ovoid  forms.  It  is  Gram  negative.  It  is  difficult  to 
demonstrate  it  in  tissues,  because  it  loses  the  stain  so  easily,  but  it 
may  be  shown  by  Klein's  method,  which  consists  in  first  staining  for 
one  minute  with  anilin  water  gentian  violet  and  then  washing  for  four 
minutes  in  iodine  iodide  of  potash  solution.  The  bacillus  does  not 
form  spores. 

Cultural  Properties. — The  organism  grows  well  on  all  of  the  ordinary 
laboratory  media.  On  gelatin  plates  it  forms  superficial,  yellowish- 
brown,  fairly  thick,  irregular  colonies  with  serrated  edges,  1  to  2  mm. 
in  diameter.  There  is  a  nipple-like  elevation  in  the  centre  of  the 
colony,  surrounded  by  a  marmorated  periphery.  The  deeper  colonies 
are  more  regularly  round  than  the  superficial  ones.  The  gelatin  is 
not  liquefied,  but  a  cloudy  halo  due  to  the  formation  of  fine  crystals 
appears  around  it  after  some  time.  In  gelatin  stick  cultures  the 
growth  is  best  at  the  surface,  rather  poor  along  the  stick  canal, 
giving  the  culture  a  nail-like  appearance.  Bouillon  is  clouded  after 
eighteen  to  twenty-four  hours,  and  a  pellicle  is  formed  on  the  surface. 
Later  a  dust-like  sediment  is  formed.  The  alkalinity  of  the  medium 
becomes  increased.  There  is  no  indol  formation.  On  agar  and 
coagulated  blood  serum  a  surface  growth  with  a  mother-of-pearl  luster 
is  formed.  It  has  been  compared,  on  account  of  the  iridescence,  to 
a  thin  film  of  petroleum  on  water.  The  addition  of  glycerin  to 
gelatin  or  agar  favors  the  growth.  On  potatoes  the  growth  of  cultures 
isolated  directly  from  lesions  caused  by  the  bacillus  leads  to  a  yellowish- 
brown,  later  a  brown  growth,  which  has  a  certain  similarity  to  the 


368          PSEUDOTUBERCULOSIS  AND  ACID-FAST  BACILLI 

growth  of  the  glanders  bacillus  on  the  same  medium.  The  bacillus  of 
pseudotuberculosis  grows  well  in  milk  without  changing  its  reaction 
or  physical  properties.  The  organism  is  not  very  resistant  to  dis- 
infectants and  is  easily  killed  by  desiccation.  It  grows  both  at  room 
and  at  incubator  temperature. 

Natural  Infection. — This  is  probably  brought  about  by  ingestion. 
The  animals  most  susceptible  to  artificial  inoculation  are  the  rabbit, 
guinea-pig,  and  mouse.  Horses,  goats,  dogs,  rats,  and  cats  are  not 
susceptible  to  the  Bacillus  pseudotuberculosis  rodentium. 

BACILLUS    PSEUDOTUBERCULOSIS   MURIUM. 

This  organism,  obtained  by  Kutscher  from  a  mouse  which  had 
died  spontaneously  with  pseudotuberculous  lesions,  is  characterized 
by  great  polymorphism.  It  forms  in  artificial  cultures  large  club- 
and  dumb-bell-shaped  individuals,  which  stain  unequally,  and  often 
closely  resemble  certain  forms  of  the  diphtheria  bacillus.  It  is  Gram 
negative.  The  bacillus  of  mouse  pseudotuberculosis  forms  on  agar 
delicate,  yellowish,  serrated  colonies,  with  short  lumpy  processes. 
It  grows  well  on  gelatin,  which  it  does  not  liquefy.  In  gelatin  stick 
cultures  a  whitish  growth,  which  sends  put  processes  into  the  mass  of 
the  culture  medium,  is  formed  along  the  canal.  It  clouds  bouillon 
in  twenty-four  to  forty-eight  hours  and  forms  a  pellicle  on  its  surface. 
It  does  not  grow  on  potatoes.  It  is  pathogenic  to  mice  if  administered 
by  inhalktion,  subcutaneously  or  intraperitoneally. 

Bongert  has  investigated  a  mouse  epizootic,  also  characterized  by 
pseudotuberculous  lesions,  and  has  isolated  from  the  latter  a  small 
bacillus  somewhat  resembling  the  pseudodiphtheria  bacillus.  He 
has  named  this  mouse  pathogenic  bacterium  Corynethrix  pseudo- 
tuberculosis  murium.  The  organism  grows  aerobically  and  anaero- 
bically  best  in  the  incubator.  It  is  1  to  2  micra  long,  0.5  micron  thick, 
and  has  a  tendency,  like  the  diphtheria  and  pseudodiphtheria  bacilli, 
to  form  pallisade-like  groups. 

BACILLUS  PSEUDOTUBERCULOSIS  OVIS. 

Occurrence. — Pseudotuberculosis  of  sheep,  or  ovine  caseous  lymph- 
adenitis, is  a  disease  which  was  first  recognized  as  something  different 
from  true  tuberculosis  by  Preisz,  whose  discovery,  however,  did  not 
attract  much  attention  at  first.  Within  a  few  years,  however,  it  was 
shown  that  this  disease  of  sheep  is  by  no  means  uncommon,  and  it 
has  since  been  encountered  extensively  in  Australia,  New  Zealand, 
Argentina,  the  United  States,  and  other  countries.  According  to 
Sivari  10  per  cent,  of  the  sheep  slaughtered  in  Buenos  Ayres  are 
infected  with  the  disease.  It  rarely  occurs  in  young  animals,  and 
when  it  does  is  limited  to  one  or  a  few  lymph  glands.  In  older 


BACILLUS  PSEUDOTUBERCULOSIS  OVIS  369 

wethers,  and  particularly  in  older  ewes,  the  fully  developed  disease 
with  extensive  lesions,  due  to  a  long  chronic  course,  is  observed. 

Pathologic  Lesions. — The  internal  organs  of  animals  which  have 
been  sick  with  this  disease  for  a  long  time  show  either  smaller  or 
larger  nodules,  inclosed  in  a  fibrous  capsule,  containing  a  caseous 
material,  greenish  yellow  in  color,  and  resembling  the  contents  of  the 
intestinal  nodules  due  to  the  intestinal  parasite  Esophagostoma 
columbianum.  The  caseous  nodules  may  reach  the  size  of  a  walnut; 
they  are  generally  found  in  the  lungs,  spleen,  and  liver,  and  more 
rarely  in  the  kidneys.  If  the  lungs  are  considerably  involved  the 
pleurae  are  thickened  and  adherent;  the  thoracic  cavity  often  contain 
a  pleuritic  exudate.  In  the  liver  numerous  small  nodules  looking 
like  miliary  tubercles  are  sometimes  found  instead  of  the  abscesses 
with  caseous  material.  According  to  Noergaard  and  Mohler,  who 
have  studied  the  disease  in  this  country,  the  principal  changes  in 
cases  not  too  advanced  are  generally  confined  to  the  lymph  glands; 
sometimes  only  a  single  gland  is  involved.  The  glands  most  com- 
monly affected  are,  in  the  order  of  their  frequency:  the  prescapular, 
precrural,  superficial  inguinal,  bronchial,  mediastinal,  sublumbar, 
deep  inguinal,  and  scrotal,  and  rarely  the  suprasternal  and  mesenteric 
glands.  Microscopic  examination  shows  that  the  caseous  mass  is 
composed  of  an  amorphous  material  surrounded  by  more  or  less 
degenerated  and  also  intact  polynuclear  leukocytes.  These  are 
again  surrounded  by  fixed  connective-tissue  cells  of  the  small  mono- 
nuclear  and  the  endothelial  type.  Giant  cells  have  never  been  found. 
The  inflammatory  cells  are  surrounded  by  fibrous  connective  tissue 
which  forms  the  limiting  capsule.  The  pseudotubercle  and  the  true 
tubercle,  according  to  Grabert,  may  be  distinguished  by  the  following 
differential  characters.  While  both  kinds  of  tubercles  lead  to  caseation, 
the  pseudotubercle  only  very  rarely  shows  calcification,  but  more 
frequently  desiccation  with  onion-like  moulding  of  the  dried  layers. 
If  pseudotuberculous  material  is  inoculated  into  the  anterior  chamber 
of  the  eye,  small  nodules  appear  after  two  to  three  days,  while  true 
tuberculosis  leads  to  nodular  formation  only  after  two  to  three  weeks. 
The  pseudotubercle  is  softer  than  the  genuine  one  and  not  grayish 
white  and  translucent  like  the  latter,  but  white  or  yellowish  and 
perfectly  opaque.  In  old  pseudotuberculous  nodules,  with  caseous 
material  young,  small  tubercles  are  not  seen  at  the  periphery  as  in 
true  tuberculosis.  Pseudotuberculosis  ovis  is  due  to  a  bacillus 
discovered  in  1891  by  Preisz  and  Guinard. 

Morphology. — The  Bacillus  pseudotuberculosis  ovis  is  a  very  small, 
slender  rod,  only  slightly  thicker  than  the  bacillus  of  hog  erysipelas. 
It  has  rounded  ends,  and  the  rods  are  from  two  to  four  times  as  wide, 
sometimes  even  longer,  and  they  vary  considerably  in  shape.  The 
ends  are  sometimes  club-shaped,  in  other  rods  they  are  pointed. 
The  bacilli  in  the  discharge  from  the  nodules  are  often  seen  in  dense 
groups,  both  inside  and  outside  of  the  cells.  In  caseous  material 
24 


370         PSEUDOTUBERCULOSIS  AND  ACID-FAST  BACILLI 

ovoid  forms  are  seen.  The  bacilli  stain  well  with  the  ordinary  watery 
anilin  stains;  they  are  Gram  positive;  they  often  do  not  stain  uniformly, 
and  then  very  much  resemble  the  diphtheria  bacillus,  but  are  smaller 
than  the  latter.  The  pseudotubercle  bacillus  of  sheep  is  not  motile 
and  does  not  form  spores.  Pure  cultures  can  best  be  obtained  from 
material  taken  from  the  outer  zone  of  the  caseous  material  in  closed 
nodules. 

Cultural  Properties. — The  bacillus  grows  poorly  at  room,  better  at 
incubator,  temperature.  The  first  generation  always  grows  poorly, 
but  the  development  is  better  in  subsequent  transplants.  The 
organism  is  a  facultative  aerobe.  On  agar  small  punctate,  grayish- 
white  colonies  appear  after  twenty-four  hours,  and  after  six  to  eight 
days  reach  their  maximum  size  of  0.5  to  3  mm.;  the  circumference 
of  the  colonies  is  serrated,  their  surface  dull  and  umbilicate,  with  con- 
centric rings  arranged  around  the  depressed  centre.  In  generations 
subsequent  to  the  first  few  the  growth  becomes  abundant,  the  colonies 
become  confluent  and  form  a  thin,  moist,  opaque,  slightly  folded 
film,  which  gives  rise  to  threads  when  touched  with  the  platinum 
loop.  In  agar  stick  cultures  small,  roundish,  grayish-white  colonies 
appear  along  the  stick  canal,  and  the  surface  becomes  covered  with  a 
growth  similar  to  that  on  agar  slants.  The  addition  of  glycerin  to 
the  agar  appears  to  be  unfavorable.  Bouillon,  during  the  first  six 
hours  of  growth,  becomes  uniformly  cloudy,  then  a  granular  sediment 
is  formed,  and  the  supernatent  fluid  becomes  clear  again;  on  the 
surface  a  dry,  grayish-white,  broken-up  pellicle  adhering  fairly 
firmly  to  the  glass  tube  at  the  margin  is  formed.  On  coagulated 
blood  serum  small,  moist,  shiny  isolated  colonies  appear  after  thirty- 
six  to  forty-eight  hours,  and  after  a  few  days  form  rhizoid  processes 
into  the  depths  of  the  medium.  The  colonies  on  horse  serum  form  a 
white  and  on  cattle  serum  an  intensely  yellow  pigment.  On  the  latter 
a  yellowish  cloudy  halo  is  formed  around  the  colonies  after  some  time. 
On  potatoes  the  bacillus  forms  a  dust-like,  dirty  white  film.  It  grows 
in  milk  and  does  not  change  its  reaction  or  physical  conditions.  It 
does  not  ferment  sugar  nor  does  it  form  phenol  or  indol.  The  bacillus 
of  Preisz  has  its  temperature  optimum  at  37°  C.,  and  growth  ceases  at 
43°  C.  The  bacillus  is  not  very  resistant  to  heat  nor  to  the  ordinary 
disinfectants. 

Inoculation  experiments  with  the  bacillus  of  Preisz  have  been 
made  by  Noergaard  and  Mohler,  and  typical  lesions  were  produced  in 
sheep  and  guinea-pigs.  Rabbits  are  more  resistant;  chickens  and 
pigeons  are  not  susceptible  to  inoculations  with  the  organism. 

An  equine  disease  known  as  Lymphangitis  ulcerosa  equorum,  or 
"Lymphangite  ulcereuse  du  Cheval"  (French),  has  been  described  in 
the  chapter  on  Glanders  under  the  head  of  Pseudoglanders.  It  is 
now  claimed  that  the  bacillus  discovered  by  Nocard  in  lesions  of 
horses  suffering  from  this  disease  is  identical  with  the  Preisz  bacillus 
of  pseudotuberculosis  of  sheep. 


ACID-FAST  BACILLI  371 


ACID-FAST  BACILLI. 

When  discussing  the  morphology  of  the  tubercle  bacillus  it  was 
pointed  out  that  it  has  peculiar  staining  properties.  While  it  is 
difficult  to  induce  the  organism  to  take  up  watery  solutions  of  basic 
anilin  stains  it  is  equally  difficult  to  decolorize  it  after  the  dye  has 
once  penetrated  into  the  substance  of  the  bacillus.  The  reason  for 
this  action  is  that  the  tubercle  bacillus  possesses  a  form  of  membrane 
composed  of  a  waxy  material.  A  few  other  bacilli  act  toward  stains 
more  or  less  like  the  tubercle  bacillus  and  are  known  under  the 
common  name  of  acid-fast  bacilli.  Several  have  been  found  living 
in  the  outside  world,  evidently  as  harmless  saprophytes. 

Moeller  has  discovered  and  described  three  varieties  of  such  bacilli. 
One  he  found  on  timothy  grass  (Phleum  arvense),  another  in  manure, 
and  a  third  in  plant  dust  in  barns.  The  three  varieties  are  easily 
raised  on  artificial  culture  media,  on  which  they  grow  rapidly,  forming 
on  the  third  or  fourth  day  a  yellowish  to  dark  orange  pigment.  The 
bacilli  are  even  more  firmly  acid-proof  than  the  tubercle  bacilli,  and 
can  still  better  withstand  dipping  in  dilute  mineral  acids  and  washing 
in  alcohol.  The  first  two  varieties  of  Moeller's  acid-fast  bacilli  grow 
best  at  temperatures  of  45°  to  50°  C.;  when  obtained  from  young 
cultures  they  show  some  motility  in  the  hanging  drop,  while  older 
cultures  display  considerable  pleomorphism  with  branching  forms. 

Moeller's  grass  bacillus  when  inoculated  intraperitoneally  into 
guinea-pigs  leads  to  pseudotubercular  lesions'- and  the  formation  of 
abscesses  containing  a  purulent  or  somewhat  caseous  material. 
Typical  tubercles  with  giant  and  epithelioid  cells  are,  however,  not 
formed.  Moeller  also  obtained  an  acid-fast  bacillus  from  a  nodule 
of  a  steer;  it  grew  very  rapidly  at  room  temperature,  and  in  intra- 
peritoneal  inoculation  produced  a  pseudotuberculosis  in  guinea-pigs. 

Petri  and  Rabinowitsch  have  isolated  from  butter,  acid-fast  bacilli 
which  closely  resemble  the  acid-fast  bacilli  of  Moeller.  Petri  found 
such  bacilli  54  times  in  102  specimens  of  butter  examined;  Rabin- 
owitsch 23  times  in  80  specimens  examined;  Klein  in  London  in  8  out 
of  100  specimens,  and  Santori  in  Rome  in  all  specimens  of  butter 
tested  for  the  presence  of  such  organisms.  The  Petri-Rabinowitsch 
bacilli  are  not  as  firmly  acid-fast  as  the  tubercle  bacillus.  When 
raised  on  agar  they  form  a  thick,  cream-like  growth,  which  later 
assumes  an  orange  color,  then  shrivels  and  becomes  cracked  and 
uneven.  If  repeatedly  passed  through  animals  the  growth  becomes 
dry  and  cracked,  and  closely  resembles  a  culture  of  tubercle  bacilli 
on  glycerin  agar.  Bouillon  cultures  of  the  Petri-Rabinowitsch  acid- 
fast  bacilli  remain  clear  and  become  covered  with  a  thick  folded 
membrane  which  gives  off  a  disagreeable  ammoniacal  odor  and  forms 
a  small  amount  of  indol.  If  these  butter  bacilli  are  inoculated  intra- 
peritoneally into  guinea-pigs  they  produce  pseudotubercular  lesions. 


372         PSEUDOTUBERCULOSIS  AND  ACID-FAST  BACILLI 

It  has  been  shown  by  experiments  made  by  investigators  upon  them- 
selves that  these  butter  bacilli  are  not  pathogenic  to  man.  Korn 
isolated  in  butter  in  Freiburg  a  bacillus  now  generally  known  as  the 
Bacillus  Freiburgensis.  It  has  nearly  the  same  staining  and  cultural 
properties  as  the  other  butter  bacilli,  and  is  pathogenic  for  white  mice 
but  not  for  guinea-pigs. 

SMEGMA  BACILLUS. 

There  occurs  on  the  human  skin,  particularly  in  the  smegma 
under  the  prepuce  and  between  the  folds  of  the  labia  majora  and 
minora  of  the  female,  an  acid-fast  bacillus  which,  in  connection  with 
human  excretions,  such  as  urine,  etc.,  may  be  confounded  with  the 
tubercle  bacillus.  This  acid-fast  bacillus  is  known  as  the  smegma 
bacillus.  According  to  Frankel  and  Neufeld  there  are  two  varieties: 
one,  called  the  "tuberculoid,"  is  more  slender  and  stains  a  bright 
scarlet  red;  the  other,  known  as  the  "diphtheroid,"  stains  more 
purplish  red,  and  is  easier  to  decolorize  than  the  former.  According 
to  Frankel  the  latter  variety  only  has  been  grown  in  artificial  cultures. 
It  develops  much  more  rapidly  than  the  tubercle  bacillus,  and  on  most 
of  the  ordinary  media.  When  these  bacilli  are  cultivated  for  several 
generations  on  artificial  media  they  lose  their  acid-fast  character,  and 
can  be  easily  decolorized. 

THE  BACILLUS  OF  LEPRA  IN  MAN  AND  RATS. 

The  human  disease  leprosy,  or  lepra,  is  very  probably  due  to  an 
acid-fast  bacillus  which  occurs  in  enormous  numbers  in  the  pathologic 
lesions  of  this  disease.  The  bacillus  has  never  been  successfully 
obtained  in  pure  cultures,  but  certain  methods  like  those  of  Weil 
and  Clegg  bring  about  a  multiplication  of  lepra  bacilli  which,  however, 
are  not  present  in  pure  cultures.  The  author  has  seen  lepra  bacilli 
multiply  by  obtaining  material  from  lepra  tubercles,  inclosing  it  in 
collodion  sacs  and  implanting  these  into  the  peritoneal  cavities  of 
monkeys,  where  they  were  left  for  several  weeks.  The  material 
became  completely  softened  under  these  conditions  and  showed 
numerous  lepra  bacilli,  which,  when  inoculated  into  other  monkeys, 
however,  failed  to  produce  the  typical  picture  of  the  disease.  Several 
French  authors,  however,  have  claimed  that  they  were  able  to  infect 
monkeys  with  human  leprosy. 

The  bacillus  of  human  leprosy  is  easier  to  stain  but  not  as  firmly  acid- 
proof  as  the  tubercle  bacillus,  and  it  can,  therefore,  be  distinguished 
from  the  latter  by  the  following  method  recommended  by  Baumgarten : 

To  a  watch-glassful  of  distilled  water  add  5  to  6  drops  of  a  saturated 
alcoholic  solution  of  fuchsin;  allow  the  cover-glass  with  the  leprous 
or  tuberculous  material  on  it  to  float  on  the  surface  of  the  stain  for 
six  or  seven  minutes.  Decolorize  one-quarter  of  a  minute  in  10  per 


PLATE  XI 


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Section  of  the  Small  Intestine  of  a  Cow  Dead  from  Chronic 
Bacterial   Dysentery.    (Johne's  Disease.) 

Oil  immersion  magnification. 


BACILLUS  OF  CHRONIC  DYSENTERY  IN  CATTLE         373 

cent,  nitric  acid  alcohol,  then  wash  in  distilled  water  and  counterstain 
with  watery  methylene  blue.  This  method  stains  the  lepra  bacillus 
red,  while  tubercle  bacilli  are  not  stained  at  all. 

Rat  Leprosy. — There  is  a  disease  in  wild  gray  rats  which  is  caused 
by  an  acid-fast  bacillus  and  which  presents  lesions  almost  identical 
with  those  found  in  human  leprosy.  The  two  diseases,  however,  are 
not  identical.  The  rat  cannot  be  infected  with  human  leprosy,  and 
there  is  no  reason  to  believe  that  the  human  disease  came  originally 
from  rats  or  that  man  can  be  infected  with  rat  leprosy. 

Occurrence. — Rat  leprosy  has  been  observed  by  Stefansky  in  Russia, 
Rabinowitsch  in  Berlin,  Dean  in  London,  Tidswell  in  Australia,  the 
English  Plague  Commission  in  India,  and  Wherry  and  McCoy  in 
California;  accordingly,  it  appears  to  occur  over  a  large  territory. 
Its  prevalence  bears  no  relation  to  human  leprosy,  as  is  shown  in 
the  Hawaiian  Islands,  where  leprosy  among  human  beings  is  common 
and  where,  in  spite  of  rewards  offered,  leprosy-infected  rats  have 
never  been  found. 

Pathology. — The  pathologic  lesions  of  this  disease  of  rats,  according 
to  Brincke"rhoff,  are  as  follows :  The  skin  in  a  well-developed  case  of 
the  disease  presents  a  patchy  alopecia  coincident  with  thickening 
and  nodule  formation  situated  in  the  subcutaneous  tissue.  The  cut 
surface  of  the  nodules  or  thickenings  is  light  yellow  in  color,  clean, 
dry,  and  cheese-like.  In  the  region  of  the  nodules  the  skin  is  atrophic, 
and  ulcers  often  form  on  the  prominent  parts  of  the  affected  area.  The 
subcutaneous  fatty  tissue  is  diminished  in  amount.  Histologically  the 
process  is  seen  to  be  practically  confined  to  the  subcutaneous  tissue 
and  to  consist  essentially  in  the  presence  of  cells  rich  in  protoplasm, 
with  vascular  nuclei,  whose  cell  body  is  more  or  less  completely 
filled  with  slender  acid-fast  bacilli.  The  subcutaneous  fat  is  replaced 
by  such  a  tissue.  Where  the  musculature  is  involved  the  muscle 
fibers  atrophy  and  the  fibers  are  infiltrated  with  the  acid-fast  bacilli. 
The  peripheral  lymph'  nodes  are  generally  involved;  they  are  enlarged 
and  on  section  opaque,  pale  yellowish-white  in  color. 

Morphology  of  Bacillus  of  Rat  Leprosy. — It  resembles  closely  the 
lepra  bacillus  of  man,  is  3  to  5  micra  long,  0.5  micron  wide;  it  is 
somewhat  more  firmly  acid-proof  than  the  human  lepra  bacillus.  It 
often  shows  a  beaded  appearance,  and  presents  itself  in  crowded 
bundles.  The  disease  can  be  transmitted  by  inoculation  to  gray  and 
white  rats.  Guinea-pigs  and  other  animals  are  not  susceptible. 


BACILLLUS  OF  CHRONIC  DYSENTERY  IN  CATTLE  (JOHNE'S 

DISEASE). 

Occurrence. — There  occurs  in  cattle  a  chronic  dysentery  with 
progressive  emaciation  and  anemia  and  finally  death,  due,  as  it 
appears,  to  an  acid-fast  bacillus  which  is  found  in  enormous  numbers 


374 


PSEUDOTUBERCULOSIS  AND  ACID-FAST  BACILLI 


FIG.  153 


in  the  dysenteric  discharges  and  in  the  mucosa  particularly  of  the 
small  intestines.  The  disease  was  first  recognized  as  one  of  a  peculiar 
type  by  Johne  and  Frothingham  in  1895.  They  found  the  acid-fast 
bacillus,  and  were  at  first  inclined  to  look  upon  it  as  an  avian  tubercle 
bacillus.  Other  cases  were  then  reported  in  Europe  by  Markus, 
Lienan  and  Van  den  Eeckhout,  Borgeaut,  and  Bang.  The  first  cases 
in  the  United  States  were  noticed  by  Pearson  in  Pennsylvania  and  by 
Beebe  in  Minnesota.  Since  then  not  a  few 
cases  have  been  observed  in  this  country. 
The  author  received  material  from  a  case  in 
1908  through  the  kindness  of  Professor  Alex- 
ander, of  Madison,  Wis.,  and  a  second  case 
likewise  from  Wisconsin,  through  Dr.  Hermann 
Schwarze. 

Pathologic  Lesions.  —  Animals  dead  from 
chronic  bacterial  dysentery  upon  postmortem 
examination  are  generally  found  to  be  very 
much  emaciated  and  extremely  anemic.  There 
are  otherwise  no  marked  changes  in  the  in- 
ternal organs  except  in  the  intestines,  which 
are  the  seat  of  characteristic  lesions.  The 
small  intestine  in  particular  shows  great  thick- 
ening. If,  however,  the  disease  has  been 
recognized  early  by  microscopic  examination 
and  inoculations  and  the  animal  has  been 
slaughtered  while  still  in  good  condition  the 
thickening  may  be  moderate;  but  if  death  has 
occurred  both  the  mucosa  and  the  submucosa 
of  the  small  intestine  are  much  thickened. 
The  former  develops  coarse,  very  prominent 
corrugations,  which  show  a  congested  and 
sometimes  even  hemorrhagic  surface.  These 
changes  may  extend  from  the  small  to  the 
large  intestines.  Microscopic  examination  of 
sections  of  the  mucosa  of  the  small  intestine 
shows  that  the  thickening  is  due  chiefly  to  the 
increase  in  fixed  connective-tissue  cells  and  to 
infiltration  by  an  edematous  exudate.  There 
are  neither  true  tubercles  nor  giant  cells,  but 
between  the  cells  enormous  numbers  of  acid- 
fast  bacilli  are  encountered.  These  are  much 
more  numerous  than  in  any  form  of  tuberculosis,  including  avian 
tuberculosis.  There  is  a  striking  similarity  in  morphology,  numbers, 
and  grouping  between  the  acid-fast  bacilli  of  this  bovine  chronic 
dysentery  and  the  bacilli  of  human  lepra. 

Morphology. — The  bacillus  of  chronic  dysentery  of  cattle  is  a  rod 
2  to  3  micra  long  and  0.5  micron  wide.    It  presents  itself  in  regular 


The  small  intestine  of  a 
cow  in  chronic  bacillary 
dysentery.  (Johne's  dis- 
ease.) 


QUESTIONS  375 

cylindrical,  in  slightly  curved,  and  in  club-shaped  forms.  It  has 
the  same  staining  properties  as  the  tubercle  bacillus,  is  acid-fast,  and 
apparently  can  well  withstand  long  washing  in  alcohol. 

Attempts  to  cultivate  the  bacillus,  as  well  as  animal  inoculations, 
have  so  far  been  unsuccessful.  Since  the  disease  always  appears  to 
lead  to  a  fatal  issue,  animals  found  to  be  infected  with  it  should  be 
slaughtered  as  soon  as  the  diagnosis  is  made.  The  differential 
diagnosis  from  intestinal  tuberculosis  can  be  made  by  the  tuberculin 
test  and  by  inoculation  experiments  upon  guinea-pigs. 


QUESTIONS. 

1.  What  is  meant  by  pseudotubercular  diseases? 

2.  Name  the  three  types  of  bacilli  which  cause  such  pseudotubercular  diseases. 

3.  Are  these  bacilli  acid-fast?     What  is  the  meaning  of  the  term  acid-fast 
as  applied  to  bacteria? 

4.  What  is  the  Bacillus  pseudotuberculosis  rodentium? 

5.  Is  it  found  as  a  saprophyte,  and  if  so,  where? 

6.  What  animals  are  susceptible  to  the  natural  infection  by  this  bacillus? 
By  what  route  does  it  generally  enter?    What  animals  are  susceptible  to  artificial 
infection  ? 

7.  Describe  the  lesions  produced  by  the  Bacillus  pseudotuberculosis  rodentium 

8.  Describe  its  morphology  and  staining  properties. 

9.  Describe  its  cultural  properties. 

10.  What  is  the  Bacillus  pseudotuberculosis  murium? 

11.  Describe  its  morphology  and  cultural  properties  and  the  lesions  which  it 
produces. 

12.  What  kind  of  disease  is  ovine  caseous  lymphadenitis  ?    Where  does  it  occur  ? 

13.  Describe  the  pathologic  lesions  of  the  disease. 

14.  What  causes  the  disease?    Who  discovered  its  specific  microorganism? 

15.  Describe  its  cultural  characteristics. 

16.  What  animals  are  susceptible  to  the  disease  (a)  under  natural  conditions; 
(6)  when  inoculated  artificially? 

17.  What  is  the  relation  between  the  bacillus  of  Preisz  (in  ovine  pseudo- 
tuberculosis)  and  that  of  Nocard  in  lymphangitis  ulcerosa  equorum? 

18.  What  are  the  three  acid-fast  bacilli  of  Moeller? 

19.  What  effect  have  these  bacilli  when  inoculated  intraperitoneally   into 
guinea-pigs  ? 

20.  What  are  the  Petri  and  the  Rabinowitsch  butter  bacilli? 

21.  Describe  their  growth  on  agar. 

22.  Describe  their  effect  in  intraperitoneal  inoculation  into  guinea-pigs. 

23.  How  can  the  characteristics  of  their  growth  on  agar  be  changed  so  that 
cultures  resemble  those  of  the  tubercle  bacillus? 

24.  What  is  the  smegma  bacillus? 

25.  How  can  the  human  lepra  bacillus  be  distinguished  from  the  human  or 
bovine  tubercle  bacilli? 

26.  Is  lepra  in  man  and  lepra  in  the  rat  identical? 

27.  Describe  the  pathologic  lesions  in  leprosy  of  rats. 

28.  Describe  the  bacillus  of  rat  leprosy. 

29.  Can  this  disease  be  transmitted  artificially,  and  to  what  animals? 

30.  Where  has  rat  leprosy  been  observed  ? 

31.  What  kind  of  disease  is  chronic  bacillary  dysentery  of  cattle?    By  what 
other  name  is  it  also  known? 

32.  Describe  its  bacillus  and  the  pathologic  lesions  produced  by  it. 

33.  Name  four  different  diseases  in  animals  due  to  different  acid-fast  bacilli. 
How  can  you  distinguish  these  four  species  by  stains,  cultural  and  inoculation 
tests? 


CHAPTER    XXXiH. 

VARIOUS  COCCI  PATHOGENIC  FOR  DOMESTIC  ANIMALS  AND  MAN 

— DIPLOCOCCUS   MENINGITIDIS   EQUI— DIPLOCOCCUS    INTRA- 

CELLULARIS— DIPLOCOCCUS  PNEUMONLE— MICROCOCCUS 

CATARRHALIS— PNEUMOCOCCUS  OF  FRIEDLANDER 

— GONOCOCCUS— MICROCOCCUS  CAPRINUS— 

MICROCOCCUS  MELITENSIS. 

INFECTIOUS  EQUINE  CEREBROSPINAL  MENINGITIS. 

Historical  and  Occurrence. — Epizootic  cerebrospinal  meningitis 
among  horses  has  evidently  been  observed  for  a  long  time.  It  was 
described  in  1813,  in  Germany,  by  Woertz,  as  "Hitzige  Kopfkrankheit" 
(febrile  disease  of  the  head).  A  more  extensive  epizootic  in  Europe 
was  described  by  Franque'  in  1824.  Large  (1847)  and  Liautard 
first  described  epizootics  in  the  United  States,  while  more  recent 
contributions  have  come  from  Wilson  and  Brimhall  and  Streit  and 
Harrison.  The  disease  was  vepy  prevalent  in  1878  and  1879  in  Saxony, 
particularly  in  the  neighborhood  of  Borna,  and  it  has  consequently 
been  occasionally  designated  as  Borna  disease. 

Pathologic  Lesions. — The  anatomic  changes  in  the  disease,  according 
to  Hutyra  and  Marek,  are  generally  not  well  marked  to  the  naked 
eye.  They  consist  in  dilatation  of  the  vessels  of  the  pia-arachnoid  of 
the  cerebrum  and  cord  and  the  presence  of  a  clear  yellowish,  not 
purulent,  exudate  in  the  ventricles.  Moore,  in  the  examination  of  a 
number  of  cases,  found  no  marked  lesions  visible  to  the  naked  eye. 
MacCallum  and  Buckley  have  found  foci  of  softening  in  the  frontal 
areas  anterior  to  the  motor  region  of  the  cortex.  It  appears,  however, 
that  the  cases  examined  by  McCarthy  and  Ravenel  were  not  of  the 
true  epidemic,  but  of  another  type.  Sometimes  a  fibropurulent 
exudate  has  been  found  at  the  base  of  the  brain  and  at  the  medulla 
oblongata. 

Bacteriology. — A  number  of  observers  have  studied  the  bacteriology 
of  the  disease.  Siedamgrotzky  and  Schlegel  have  found  a  coccus 
which  generally  presents  itself  singly  and  more  rarely  as  a  diplococcus. 
It  is  Gram  positive.  On  gelatin  plates  it  forms  dirty,  grayish-white, 
well-defined  colonies,  with  a  denser  centre;  on  agar,  white, 
shining,  sharply  defined  colonies.  The  organism  clouds  bouillon. 
Johne  found  a  coccus  which,  while  quite  similar  to  the  one  just  de- 
scribed, varied  from  it  in  some  details,  and  was  often  seen  in  short 
chains  and  intracellularly  in  leukocytes.  It  was,  therefore,  much 
like  the  Diplococcus  intracellularis  of  Jaeger- Weichselbaum,  which 


DIPLOCOCCUS  INTRACELLULARIS  MENINGITIDIS         377 

causes  epidemic  cerebrospinal  meningitis  in  man.  Ostertag  found 
cocci  very  much  like  those  of  Johne;  they  are  Gram  negative  and 
closely  resemble  the  organism  causing  the  human  disease.  The 
organism  of  Ostertag,  when  inoculated  subdurally  into  horses, 
produced  a  more  or  less  typical  attack  of  cerebrospinal  meningitis. 
Wilson  and  Brimhall,  in  cases  of  cerebrospinal  meningitis  in  horses 
and  other  domestic  animals,  found  both  cocci  of  the  type  of  the 
Jaeger- Weichselbaum  organism  and  of  FrankePs  pneumococcus.  It 
appears,  therefore,  that  cerebrospinal  meningitis  in  the  horse,  like 
cerebrospinal  meningitis  in  man,  may  be  caused  by  more  than  one 
bacterium;  the  etiology,  however,  of  the  disease  in  equines  is  not 
yet  satisfactorily  cleared  up  and  further  investigations  are  necessary. 

Cerebrospinal  meningitis  has  also  been  occasionally  found  in 
cattle,  sheep,  and  swine.  The  description  of  the  disease  in  these 
animals  is  still  very  fragmentary. 

Johne  has  named  his  organism  Diplococcus  intracellularis  equi. 


DIPLOGOGGUS  INTRACELLULARIS  MENINGITIDIS. 

This  organism,  first  seen  by  Weichselbaum  and  later  carefully 
studied  by  Jaeger,  is  generally  the  cause  of  cerebrospinal  meningitis 
in-  man.  It  is  a  biscuit-shaped  diplococcus,  and  frequently  occurs 
in  large  numbers  in  the  protoplasm  of  pus  corpuscles  of  the  fibrino- 
purulent  exudate  found  at  the  convexity  and  at  the  base  of  the  brain 
in  human  cerebrospinal  meningitis.  The  diplococcus  stains  well  with 
the  ordinary  watery  anilin  stains,  but  is  easily  decolorized  when 
treated  by  Gram's  method.  Pure  cultures  can  generally  be  obtained 
from  cerebrospinal  fluid,  drawn  with  aseptic  precautions.  Of  this 
fluid  1  to  2  c.c.  is  inoculated  into  blood  serum  or  glycerin-agar  tubes. 
Pus  in  cerebrospinal  meningitis  can  be  obtained  by  spinal  puncture. 
This  consists  in  the  introduction  of  a  strong  hypodermic  needle, 
4  to  8  cm.  long,  attached  to  an  all-glass  (Luer)  large  hypodermic 
syringe,  into  the  spinal  canal,  about  1  cm.  from  the  middle  line  and 
between  the  third  and  fourth  lumbar  vertebrae.  The  diplococci  of 
cerebrospinal  meningitis  grow  in  the  incubator  at  blood  temperature 
only.  They  develop  around  surface  colonies  with  an  opaque  yellowish- 
brown  centre  having  a  flat,  thin  periphery;  the  edges  may  be  circular 
and  straight  or  dentate.  Subcutaneous  inoculations  into  laboratory 
animals  are  generally  not  pathogenic  for  them,  but  intraperitoneal  and 
intravenous  injections  often  cause  a  fatal  result  in  mice  and  rabbits. 
Flexner  and  Joblin  have  prepared  an  antimeningitis  serum  in  the 
horse  which  has  proved  an  excellent  therapeutic  agent  in  the  human 
disease,  but  it  must  be  injected  into  the  spinal  canal  in  comparatively 
large  doses  (15  to  30  c.c.)  often  repeatedly,  and  it  is  necessary  each 
time  first  to  withdraw  an  equivalent  or  larger  amount  of  the  purulent 
cerebrospinal  fluid. 


378     COCCI   PATHOGENIC  FOR    DOMESTIC    ANIMALS  AND  MAN 


DIPLOCOCCUS  PNEUMONIA  OF  FRANKEL-WEICHSELBAUM. 

In  a  great  majority  of  cases  this  organism  is  the  cause  of  croupous 
or  lobar  pneumonia  in  man ;  it  also  causes  inflammatory  and  suppu- 
rative  processes  in  the  middle  ear,  the  joints,  the  pleura,  the  peri-  and 
endocardium,  and  sometimes  cerebrospinal  meningitis.  It  is  also 
called  Diplococcus  lanceolatus,  and  was  probably  first  seen  by  Sternberg 
and  Pasteur.  The  typical  shape  of  the  organism,  when  seen  singly 
and  in  pairs,  is  the  elongated  coccus.  The  diplococci  show  par- 
ticularly often  in  the  shape  of  candle  lights,  hence  the  name  Diplo- 
coccus lanceolatus,  which  means  lancet-shaped.  In  pathologic 
exudates  the  organism  frequently  appears  in  short  chains  in  which 
the  subdivision  into  diplococci  is  generally  well  marked,  and  it  almost 
invariably  shows  a  distinct,  quite  large  capsule,  because  of  which  it  is 
known  also  as  the  capsule  coccus  of  pneumonia.  As  a  rule,  the  capsule 
is  not  shown  on  artificial  culture  media,  but  sometimes  it  appears  on 
sterile  sputum  or  in  sterile  fluid  blood  serum  or  milk.  The  diplo- 
coccus  of  pneumonia  does  not  form  spores,  is  not  motile,  and  possesses 
no  flagella.  It  is  Gram  positive.  The  organism,  after  a  number  of 
transplants,  often  forms  longer  chains,  and  the  individual  cocci 
become  perfectly  round  and  lose  the  lancet-shape  entirely.  It  grows 
rather  poorly  on  artificial  culture  media  and  only  at  blood  temperature, 
both  aerobically  and  anaerobically.  The  superficial  cultures  on  agar 
are  thin  films,  quite  transparent.  The  deeper  colonies  are  very  small, 
and  when  examined  with  a  low  power  of  the  microscope  appear  light 
yellow  or  light  brown,  and  finely  granular.  The  growth  on  gelatin 
plates  kept  at  24°  C.  is  scanty  and  poor.  In  agar  stick  cultures  there 
is  very  little  growth  on  the  surface,  but  more  along  the  stick  where  a 
band-like  strip  is  formed.  On  agar  or  blood-serum  slants  a  fine  veil 
composed  of  small  dewdrop-like  colonies  is  formed.  Nutrient 
bouillon  and  fluid  blood  serum  become  cloudy,  and  a  scanty,  loose, 
whitish  sediment  is  formed  at  the  bottom.  The  growth  on  potatoes  is 
practically  invisible,  and  is  only  revealed  by  microscopic  examination 
of  material  taken  from  the  surface.  Milk  becomes  coagulated. 
The  addition  of  glycerin  and  glucose  to  the  media  is  favorable  to  the 
growth.  The  reaction  of  the  culture  soils  must  be  faintly  alkaline. 
The  best  medium  is  a  serum  agar  prepared  of  two  parts  of  agar  to  which 
one  part  of  human-blood  serum  has  been  added.  The  organism 
generally  dies  out  rapidly  in  artificial  cultures.  Sometimes  a  second 
generation  fails.  It  is  always  necessary  to  make  frequent  transplants. 
The  coccus  perishes  so  quickly  in  pure  cultures  because  it  rapidly 
forms  acid  in  the  presence  of  which  it  cannot  live.  The  virulency 
shown  by  different  stems  in  first  cultures  varies  greatly;  it  is  lost 
rapidly  in  subsequent  transplants.  It  appears  from  Rosenou's  wrork 
that  virulent  diplococci  are  unaffected  by  phagocytosis,  avirulent  types 
alone  are  engulfed  and  destroyed  by  leukocytes.  While  pneumonia 


PNEUMOCOCCUS  OF  FRIEDLANDER  379 

in  man  is  in  a  majority  of  cases  caused  by  the  Diplococcus  lanceolatus, 
it  may  also  be  caused  by  the  Diplococcus  or  Diplobacillus  of  Fried- 
lander,  the  Micrococcus  catarrhalis,  and  the  bacilli  of  influenza, 
glanders,  diphtheria,  plague,  and  others. 

Whether  every  case  of  contagious  pneumonia  of  horses  is  always 
due  to  equine  influenza,  i.  e.,  to  the  Bacillus  bipolaris  equisepticus,1 
or  whether  certain  non-contagious  pneumonias  in  horses  are  due  to 
other  bacteria  is  a  question  on  which  opinion  is  divided.  It,  however, 
appears  established  that  the  diplococcus  pneumonise  or  lanceolatus 
is  also  the  cause  of  pneumonia  in  the  horse,  and  that  in  old  animals, 
or  after  long-continued  debilitating  diseases  it  frequently  leads  to  a 
fatal  issue. 

MIGROGOCGUS  GATARRHALIS. 

This  was  first  seen  by  Seiffert  and  obtained  in  pure  cultures  by 
Kirchner  and  by  Pfeiffer.  It  is  a  biscuit-shaped  coccus,  generally 
occurring  in  pairs  or  tetrads.  It  stains  well  with  the  ordinary  watery 
anilin  stains,  but  is  Gram  negative.  It  is  not  motile,  and  does  not 
form  spores.  It  is  found  in  the  normal  mucous  membranes,  and  also 
in  diseases  of  the  upper  or  lower  respiratory  tract  in  man.  It  grows 
on  the  ordinary  laboratory  culture  media  between  20°  and  40°  C.,  best 
at  blood  temperature.  It  forms  grayish-white  or  yellowish-white 
colonies  with  irregular  margins,  as  if  artificially  cut  out.  Gelatin  is 
not  liquefied;  bouillon  becomes  cloudy,  a  pellicle  often  forming  on  the 
surface.  Milk  is  not  coagulated.  The  organism  varies  considerably 
in  virulency.  It  sometimes  kills  guinea-pigs,  rabbits,  and  mice  in 
intraperitoneal  inoculations. 


PNEUMOCOCCUS  OF  FRIEDLANDER. 

This  organism  should  more  properly  be  called  the  Pneumobacillus 
of  Friedldnder,  as  it  is  not  a  true  coccus  but  a  short  bacillus.  In 
pathogenic  exudates  it  is  often  so  short  that  it  was  at  first  mistaken 
for  a  coccus.  It  generally  occurs  in  groups  of  two  or  in  tetrad  form 
or  in  short  chains,  and  when  found  in  sputum  or  pus  frequently 
possesses  a  capsule.  It  can  easily  be  differentiated  from  the  Diplo- 
coccus pneumonia  of  Frankel  because  it  is  Gram  negative.  It  does 
not  form  spores,  is  not  motile,  and  possesses  no  flagella.  It  is  aerobic. 
It  shows  a  nail  growth  in  gelatin  stick  culture,  i.  e.,  it  forms  a  heavy 
growth  on  the  surface  from  which  a  streak  of  culture  extends  along 
the  stab.  It  does  not  liquefy  the  gelatin,  but  imparts  to  it  a  distinct 
brownish  tint  distinguishing  it  from  the  very  similar  growth  which 
the  Bacillus  aerogenes  forms  on  this  medium.  The  growth  on 

1  Described  in  the  chapter  on  the  Hemorrhagic  Septicemia  Bacilli. 


380     COCCI  PATHOGENIC   FOR   DOMESTIC   ANIMALS   AND  MAN 

gelatin  plates  at  the  end  of  twenty-four  hours  shows  small,  white, 
circular  colonies,  which  increase  rapidly  in  size.  The  colonies  on 
agar  are  grayish  white  and  moist.  On  blood  serum  slants  an  abundant 
grayish-white,  moist,  and  shiny  growth  is  formed,  and  on  potatoes  a 
thick,  yellowish- white  shining  layer.  Milk  is  not  coagulated.  Lactose 
and  glucose  are  fermented  with  the  formation  of  acid.  The  diplo- 
bacillus  of  pneumonia  has  not  been  encountered  as  the  natural  cause 
of  disease  in  domestic  animals. 


GONOCOCCUS. 

The  gonococcus,  or  Micrococcus  gonorrhoeae,  is  an  organism 
occurring  in  pairs  and  occasionally  in  tetrads.  The  individual  cocci 
forming  the  pair  are  flattened  at  their  opposing  surfaces,  often  some- 
what resembling  a  kidney  or  coffee  bean  in  form.  The  organism  is  the 
cause  of  gonorrheal  inflammation  of  the  urethra  in  men  and  women. 
It  also  penetrates  deeper  into  the  genito-urinary  tract,  and  causes 
epididymitis,  ovarian  abscess,  salpingitis,  inflammation  of  the  pelves 
of  the  kidneys  (pyelonephritis),  peritonitis,  inflammation  of  the 
joints,  and  endocarditis.  It  is  generally  spread  in  sexual  intercourse. 
It  may  also  be  transferred  with  the  fingers  or  otherwise  to  the 
eye  and  cause  a  very  dangerous  conjunctivitis.  The  organism  in 
pus  is  frequently  found  intracellularly  in  the  protoplasm  of  the 
leukocytes.  It  stains  very  rapidly  with  the  watery  anilin  stains,  but 
also  loses  the  stains  very  easily,  and  is  Gram  negative.  It  is  difficult 
to  grow  on  artificial  culture  media.  The  best  medium  is  an  ordinary 
agar,  two  parts,  to  which  one  part  of  sterile  human-blood  serum1  has 
been  added.  The  ingredients  are  mixed  after  the  melting  of  the  agar 
and  again  solidified  after  inoculation  from  fresh  gonorrheal  pus. 
Gonococci  form  colonies  on  such  serum  agar  after  sixteen  to  twenty- 
hours,  but  they  are  so  small  as  to  be  only  visible  with  a  good  hand 
magnifying  glass  or  the  low  power  of  the  microscope.  After  twenty- 
four  hours  the  colonies  have  attained  the  size  of  a  pinhead  and  do  not 
grow  much  larger.  They  are  light  gray,  translucent,  and  of  a  peculiar 
tenacious,  slimy  consistency.  They  generally  remain  round,  well 
defined,  and  do  not,  as  a  rule,  become  confluent,  but  may  touch  each 
other.  The  culture  soil  in  transmitted  light  somewhat  resembles  the 
surface  of  ice  which  has  been  much  cracked.  In  exceptional  cases  the 
colonies  become  confluent  and  form  grayish-white  films.  Colonies 
of  gonococci  closely  resemble  colonies  of  the  Diplococcus  lanceolatus, 
but  the  former  are  generally  somewhat  larger,  less  translucent,  and 
more  highly  colored.  Gonorrhea  is  a  strictly  human  disease.  It  does 
not  occur  naturally  among  animals,  and  even  artificial  inoculation  is 
very  difficult  and  rarely  successful.  Mice  in  intraperitoneal  infection 
develop  a  localized  peritonitis. 

1  Ascitic,  hydrocele,  or  pleuritic  fluid  may  be  used  in  place  of  the  human-blood  serum  in 
the  preparation  of  the  medium. 


MALTA  FEVER  AND  THE  MICROCOCCUS  MELITENSIS     381 


MICROCOCCUS  CAPRINUS. 

Under  this  name  Mohler  and  Washburn  have  described  a  coccus 
which  is  claimed  to  be  the  cause  of  takosis  (i.  e.,  a  wasting  disease  of 
Angora  goats).  The  most  characteristic  morbid  lesions  of  this  affec- 
tion are  emaciation  and  anemia.  Consolidated  areas  are  generally 
found  in  the  lungs,  the  myocardium  is  pale,  soft,  and  flabby,  and  the 
epicardium  and  endocardium  may  present  hemorrhagic  spots.  The 
kidneys  are  anemic  and  softened  and  the  spleen  is  small  and  may  be 
adherent  to  the  neighboring  structures.  The  Micrococcus  caprinus 
has  been  isolated  from  the  heart's  blood.  It  usually  presents  itself  in 
pairs,  and  is  pathogenic  for  goats,  chickens,  rabbits,  guinea-pigs,  and 
white  mice,  but  not  for  sheep,  dogs,  or  rats. 


MALTA  FEVER  AND  THE  MICROCOCCUS  MELITENSIS. 

Occurrence  and  Historical. — Malta  or  Mediterranean  fever  is  a 
septicemic  disease  of  man,  caused  by  the  Micrococcus  melitensis, 
discovered  by  Bruce  in  1887.  It  is,  however,  by  no  means  confined 
to  Malta,  as  cases  have  been  reported  from  Greece,  Italy,  Spain, 
Turkey,  North  and  South  Africa,  America,  India,  and  the  Philippine 
Islands.  The  affection  is  of  particular  interest  to  the  veterinarian 
because  it  is  primarily  a  disease  of  goats  and  is  transferred  through 
their  raw  milk  to  man. 

Morphology. — The  Micrococcus  melitensis  is  a  very  small  coccus, 
measuring  about  0.4  by  0.3  micron.  It  is  seen  as  a  monococcus  or  as 
diplococci,  more  rarely  in  short  chains.  The  diameter  of  the  individual 
stained  cocci,  according  to  Bruce,  is  about  0.33  micron.  Longer 
chains  of  10  to  14  cocci  are  frequently  seen  in  older  bouillon  cultures. 
The  union  of  the  individual  links  is  evidently  not  very  firm  and  the 
chains  in  stained  preparations  are  broken  up  into  smaller  segments. 
Involution  forms,  which  come  to  resemble  bacilli,  are  found  in  older 
cultures.  The  organism  does  not  form  spores,  is  not  motile,  and  has 
no  flagella.  It  stains  well  with  the  ordinary  anilin  dyes,  and  is  Gram 
negative. 

Cultural  Properties. — The  organism  grows  best  in  culture  media 
which  are  slightly  acid  to  phenolphthalein  +  1  (alkaline  to  litmus). 
It  grows  best  at  blood  temperature,  but  has  a  wide  range  of  temper- 
ature. The  growth  is  slow,  and  it  generally  requires  three  to  four  days 
before  colonies  can  be  seen  with  the  naked  eye.  The  surface  of 
agar  plates  liberally  inoculated  after  twenty-four  to  thirty-six  hours 
somewhat  resembles  ground  glass  under  a  low  power  of  the  micro- 
scope. After  twenty-four  to  seventy-two  hours  individual  dewdrop-like 
colonies  are  visible.  In  about  eight  days  they  reach  a  diameter  of 
1.5  mm.  They  are  round,  regularly  spherical;  the  superficial  colonies 


382     COCCI  PATHOGENIC   FOR    DOMESTIC    ANIMALS    AND  MAN 

are  convex,  with  a  darker  centre.  The  deep  colonies  are  biconvex, 
with  a  smooth  margin.  According  to  Eyre  the  best  culture  medium 
is  litmus  nutrose  agar,  prepared  with  cattle  serum.  The  reaction  on 
this  medium  is  not  changed  and  the  litmus  preserves  its  bluish  color. 
In  fluid  serum  the  coccus  forms  a  fine  flocculent  precipitate;  the 
supernatant  fluid  generally  remains  clear;  a  clouding  occurs  only 
rarely.  The  growth  on  gelatin  is  quite  slow.  Liquefaction  does 
not  occur.  Nutrient  bouillon  after  seventy-two  hours  becomes 
uniformly  cloudy,  but  after  eight  days  a  sediment  begins  to  form,  and 
after  four  weeks  the  entire  growth  has  sunk  to  the  bottom  and  the 
supernatant  fluid  is  again  clear.  The  growth  on  potatoes  is  very 
^canty.  Sugars  are  not  fermented.  The  organism  is  easily  destroyed 
by  moist  heat,  less  easily  by  dry  heat.  It  can  withstand  drying  out 
for  a  considerable  time,  but  is  easily  killed  by  direct  sunlight,  less 
easily  by  diffuse  daylight.  The  organism  is  pathogenic  in  artificial 
inoculation  to  monkeys,  rodents,  equine,  cattle,  sheep,  and  goats.  The 
Mediterranean  Fever  Commission  found  the  organism  in  the  milk 
of  goats  on  Malta,  and  the  blood  of  such  animals  exhibited  specific 
agglutinative  power  for  the  coccus.  Goats  caii  be  infected  by  feeding 
and  subcutaneous  and  intravenous  injections,  and  the  organism  can 
afterward  be  recovered  in  the  milk.  Anderson,  1905,  reports  an 
outbreak  of  Malta  fever  in  a  crew  of  a  vessel  who  had  been  consuming 
milk  from  goats  bought  in  Malta  for  importation  into  the  United 
States.  Examination  of  some  of  the  goat's  milk  showed  infection 
with  the  Micrococcus  melitensis. 


QUESTIONS. 

1.  Where  has  epidemic  or  infectious  equine  cerebrospinal   meningitis  been 
observed  ? 

2.  What  organisms  have  been  held  to  be  the  etiologic  factor  in  this  disease? 

3.  Describe  the  pathologic  lesions  of  the  disease. 

4.  What  are  the  views  as  to  the  relation  of  the  Diplococcus  intracellularis 
equi  and  the  Diplococcus  intracellularis  of  Weichselbaum  ? 

5.  How  can  the  latter  be  obtained  in  pure  culture? 

6.  Describe  its  morphology  and  cultural  characteristics. 

7.  To  what  organism  is  human  pneumonia  generally  due? 

8.  Describe  its  morphology  and  cultural  properties. 

9.  In  what  other  human  diseases  has  it  been  found? 

10.  What  is  most  commonly  the  cause  of  pneumonia  in  the  horse  ? 

11.  Does  the  Diplococcus  lanceolatus  ever  cause  pneumonia  in  the  horse? 

12.  Describe   the   Micrococcus   catarrhalis.     Where   found?     Does   it   cause 
disease  ? 

13.  Describe  the  pneumococcus  of  Friedlander.    Is  it  Gram  +  or  —  ? 

14.  Describe  the  gonococcus.    What  animal  diseases  does  it  cause? 

15.  How  can  it  be  obtained  in  pure  cultures? 

16.  What  is  takosis  in  goats?    What  organism  causes  it? 

17.  What  is  Malta  or  Mediterranean  fever? 

18.  What  organism  causes  it? 

19.  Describe  its  morphologic  and  cultural  properties. 

20.  How  contracted  by  man  ? 


CHAPTEK    XXXIII. 

SPIRILLA— PATHOGENIC  VIBRIONES— SPIROCHETE— THE  VIB- 

RIONES  OF  CHICKEN  SEPTICEMIA  AND  OF  ASIATIC 

CHOLERA— SPIROCHETE  IN  MAN,  OTHER 

MAMMALS  AND  BIRDS. 

The  Systematic  Classification  of  Spiral  Bacteria. — The  spiral  bacteria 
may  be  divided  into  two  groups.  One  comprises  curved,  rod-like 
organisms  shaped  very  much  like  a  comma.  They  form  a  real  spiral 
only  when  a  number  adhere  together.  The  other  group  is  composed 
of  organisms  which  are  true  spirals,  their  bodies  representing  wavy 
filaments  which  may  best  be  likened  to  corkscrews  or  winding 
stairways.  Members  of  the  first  group  are  designated  as  Vibrio  (pi. 
vibriones) ;  members  of  the  second  as  spirillum  (pi.  spirilla)  or  spiro- 
cheta  (pi.  spirochete).  The  term  spirillum,  however,  is  not  used  in  a 
very  strict  manner  and  the  comma-shaped  organism  of  Asiatic  cholera 
is  known  both  as  the  vibrio  and  the  spirillum  of  Asiatic  cholera,  while 
in  accord  with  strict  nomenclature  it  should  be  known  as  a  vibrio 
exclusively.  The  organisms  of  the  type  of  vibrio  or  spirillum  are 
generally  very  lively  motile.  The  motility  of  vibriones  does  not  differ 
much  from  that  of  lively  motile  bacilli,  but  the  motion  of  the  spiro- 
chete is  peculiar  and  characteristic.  It  consists  in  a  rotation  around 
their  long  axis,  forward  and  backward  like  a  spiral  spring  released 
from  compression,  and  a  bending  motion  of  the  whole  body.  The 
vibriones  generally  possess  a  single  flagellum  at  one  end,  but  flagella  at 
both  ends  or  multiple  flagella  at  one  or  both  ends  are  also  encountered. 
Spirilla  may  have  one  flagellum  at  one  end,  one  at  either  end,  several 
flagella  at  one  end,  or  flagella  surrounding  the  entire  body.  There 
has  never  been  any  doubt  in  regard  to  the  classification  of  the  vibriones 
among  the  bacteria,  i.  e.,  among  the  vegetable  microorganisms. 
Recently  however,  it  has  been  claimed,  particularly  by  Schaudin  and 
others,  that  the  spirochete  are  protozoa  and  near  relatives  of  the 
trvpanosomes,  if  not  indeed  themselves  true  flagellata  of  this  type. 
The  question  will  be  considered  fully  at  the  end  of  this  chapter.  Most 
vibriones  are  harmless  saprophytes,  but  two  species  at  least  possess 
very  pathogenic  properties. 


VIBRIONES. 

Vibrio  Metchnikovi,  or  Spirillum  of  Metchnikoff . — In  the  investigation 
in  1887  of  an  epizootic  among  chickens  in  Odessa,  Gamaleia  dis- 


384        SPIRILLA,  PATHOGENIC  VIBRIONES,  SPIROCHETE 


FIG.  154 


covered  as  its  cause  a  comma-shaped  bacterium  which  he  named  in 
honor  of  Metchnikoff.  The  disease  is  generally  fatal  after  forty- 
eight  hours,  and  its  characterized  by  violent  diarrhea  without  fever. 
Postmortem  examination  shows  the  intestinal  mucosa  inflamed,  its 
epithelium  desquamated;  the  contents  of  the  intestines  are  fluid  and 
mixed  with  blood.  The  fluid  contents  contain  enormous  numbers  of 
comma-shaped  bacteria,  which  very  much  resemble  the  spirillum  of 
Asiatic  cholera  in  man  (see  below).  For  this  reason  the  disease  has 
been  called  vibrio  cholera  or  vibrio  septicemia  of  chickens.  The 
spirillum  of  Metchnikoff,  as  found  in  the  intestines  of  dead  chickens, 
occurs,  in  addition  to  the  typical  comma  shape,  also  in  shorter, 
thicker  specimens,  which  look  almost  like  cocci.  The  vibrio  is  lively, 
motile,  and  possesses  one  long  slender  flagellum.  The  organism 
forms  longer  spirals  in  older  cultures  in  which  the  individual  commas 

adhere  to  each  other  in  chains. 
The  spirillum  of  Metchnikoff 
stains  well  with  watery  fuchsin, 
and  is  Gram  negative.  It  grows 
well  in  the  ordinary  laboratory 
media,  and  the  cultures  closely 
resemble  those  of  the  spirillum 
of  Asiatic  cholera.  The  vibrio 
of  chicken  septicemia  grows 
rapidly  on  gelatin  plates  at  room 
temperature,  and  after  twenty- 
four  to  thirty  hours  has  formed 
pinhead-sized  circular  colonies, 
which  rapidly  liquefy  the  me- 
dium. The  colonies  under  a  low 
power  of  the  microscope  appear 
granular  and  yellowish  or  brown- 
ish. Older  cultures  which  have 
been  transplanted  for  a  long 
time  on  artificial  media  do  not  liquefy  gelatin  as  rapidly  as  cultures 
recently  isolated  from  infected  animals.  The  growth  in  gelatin  stick 
cultures  is  much  more  rapid  than  that  of  the  vibrio  of  Asiatic  cholera. 
Bouillon  is  rapidly  clouded  and  a  fairly  strong  white  pellicle  forms  on 
the  surface  of  the  medium.  On  agar  the  vibrio  forms  a  yellowish,  on 
potatoes  a  yellowish-brown,  growth.  The  vibrio  of  Metchnikoff  cannot 
be  safely  distinguished  by  any  of  its  cultural  characteristics  from  the 
vibrio  of  Asiatic  cholera.  Both  also  form  indol  and  nitrites  in  bouillon, 
and  these  upon  the  addition  of  a  few  drops  of  chemically  pure  sulphuric 
acid  give  a  red  color  reaction.  The  differentiation  between  the  two 
organisms,  however,  can  be  made  by  animal  experiments.  The 
vibrio  of  Metchnikoff  kills  pigeons  if  injected  into  the  thoracic  muscles 
within  twenty-four  hours.  Postmortem  examination  of  the  dead  birds 
shows  the  injected  muscle  swollen,  yellowish  in  color,  and  infiltrated, 


Spirillum  of  Asiatic  cholera  smear  from  the 
intestines  of  a  man  dead  from  cholera.  X  1000. 
(Author's  preparation.) 


VIBRIONES  385 

with  a  serous  fluid  which  contains  innumerable  spirilla.  The  blood 
also  contains  many  spirilla.  The  mucosa  of  the  intestines  is  pale  and 
the  intestinal  contents  are  liquid  and  likewise  contain  the  organism. 
Guinea-pigs  also  generally  succumb  to  subcutaneous  infection  within 
twenty-four  hours.  A  hemorrhagic  edema  is  formed  at  the  place  of 
inoculation,  and  the  fluid  and  blood  show  enormous  numbers  of 
vibriones.  The  vibrio  of  Asiatic  cholera,  on  the  contrary,  in  sub- 
cutaneous or  intravascular  injection  never  produces  a  rapidly  fatal 
septicemia  in  these  animals,  which  are  so  susceptible  to  the  spirillum 
of  Metchnikoff.  In  spite  of  their  great  morphologic  and  cultural 
similarities  the  two  organisms  are  by  no  means  identical.  They  are 
very  different  in  their  pathogenicity  and  animals  cannot  be  immunized 
with  one  species  against  the  other. 

Fio.  155 


A  characteristic  series  of  cholera  cultures  in  gelatin;  from  right  to  left,  one,  two,  three,  four, 
and  six  days'  growth.     (Dunham.) 

Vibrio  of  Asiatic  Cholera. — This  organism  was  discovered  by  Robert 
Koch  in  India.  Asiatic  cholera  is  a  disease  of  man  and  does  not 
naturally  occur  among  the  lower  animals.  The  disease  is  endemic 
in  India,  but  has  spread  from  there  on  various  occasions  into  Europe, 
America,  and  other  countries.  It  is  one  of  the  most  fatal  epidemics  of 
mankind,  and  has  a  very  high  mortality.  The  great  similarity  between 
the  spirillum  of  Asiatic  cholera  and  the  Vibrio  Metchnikovi  has  already 
been  referred  to  above;  it  also  closely  resembles  several  other  spirilla, 
which  will  be  described  later. 

The  cholera  vibrio,  also  called  the  comma  bacillus  of  Koch,  is  a 
curved  organism,  and  in  artificial  cultures  often  forms  longer  spirals 
25 


386         SPIRILLA,  PATHOGENIC  VIBRIONES,  SPIROCHETE 

composed  of  individual  commas  adhering  together.  It  is  actively 
motile,  possesses  one  flagellum,  and  has,  like  all  the  organisms  of  this 
group,  the  same  staining  properties  as  the  vibrio  of  Metchnikoff.  In 
older  cultures  very  irregular  involution  forms  are  seen.  Pure  cultures 
of  the  cholera  spirillum  can  best  be  obtained  by  mixing  fecal  matter 
containing  it  with  nutrient  bouillon  in  a  flask.  When  kept  at  blood 
temperature  in  the  incubator  a  white  pellicle  forms  on  the  surface. 
This  is  chiefly  composed  of  rapidly  growing  vibriones,  and  if  tubes  are 
inoculated  from  the  pellicle,  and  gelatin  plates  poured,  pure  cultures 
can  be  obtained.  In  the  lower  stratum  of  the  gelatin  plates  small 
white  dots  appear,  which  grow  up  to  the  surface  and  liquefy  the 
medium.  The  color  of  the  colonies  soon  -turns  yellowish.  In  gelatin 
stick  cultures  the  liquefaction  leads  to  a  funnel-shaped  excavation 
of  the  surface.  The  organism  grows  in  milk,  which  it  does  not  visibly 
change,  but  under  natural  conditions  it  is  soon  killed  in  milk  because 
it  is  very  sensitive  to  acid. 

Vibrio  Proteus,1  or  the  Spirillum  of  Finkler  and  Prior. — This  organism 
was  discovered  by  Finkler  and  Prior  in  the  feces  of  persons  sick  with 
diarrhea.  The  spirilla  are  curved  and  somewhat  longer  and  coarser 
than  the  cholera  vibrio ;  they  are  often  pointed  at  the  ends  and  thicker 
in  the  middle.  In  artificial  cultures  they  form  spirals  which  generally 
are  not  as  long  as  those  of  the  two  preceding  vibriones.  When  the 
culture  medium  is  not  very  favorable  the  spirilla  vary  greatly  in  shape, 
and  often  form  large  oval  bodies  or  very  coarse  curved  bacilli.  For 
this  reason  the  organism  has  been  called  Vibrio  proteus  by  Buchner. 
The  colonies  on  gelatin  are  darker  and  more  regularly  circular  than 
those  of  the  cholera  vibrio.  The  liquefaction  in  gelatin  stick  cultures 
is  very  energetic,  and  progresses  to  a  sacculate  zone  within  forty-eight 
hours.  The  organism  ferments  sugar  with  the  formation  of  acid  and 
produces  a  fetid  smell  in  all  culture  media.  The  spirillum  of  Finkler 
and  Prior  is  probably  not  pathogenic  to  man,  but  only  a  harmless 
saprophyte  occasionally  found  in  the  intestinal  tract.  It  is  very 
slightly  pathogenic  to  animals  in  subcutaneous  and  intraperitoneal 
injection. 

Spirillum  of  Denecke,  or  Vibrio  Tyrogenum. — This  organism  belongs 
to  this  group.  It  is  not  pathogenic,  and  was  first  found  in  old  cheese 
by  the  author  whose  name  it  bears.  It  is  somewhat  smaller  than 
the  vibrio  of  cholera,  and  in  artificial  cultures  forms  long,  slender 
spirilla. 

Water  Spirilla. — A  number  of  spirilla  of  this  group  have  been  found 
in  the  water  of  rivers  in  Europe  and  America,  such  as  the  Vibrio 
Berolinensis  (found  in  Berlin),  the  Vibrio  Danubius  (found  in  the 
Danube),  the  Vibrio  Schuylkiliensis  (found  in  Philadelphia  by  Abbott). 
They  are  all  non-pathogenic. 


1  The  student  must  not  confound  the  Vibrio  proteus  with  the  Bacillus  proteus,  which  is 
an  entirely  different  organism. 


SPIROCHETE  387 


SPIROGHETE. 

The  first  spirochete  described  under  the  name  of  Spirocheta 
plicatilis  by  Ehrenberg  and  found  in  marshy  water  probably  does 
not  belong  to  the  organisms  classified  today  as  spirilla,  or  spirochete. 
The  most  important  pathogenic  species  are  the  following :  Spirocheta 
Obermeieri,  found  in  relapsing  fever  in  man ;  Spirocheta  Duttoni,  found 
in  African  tick  fever  in  man,  and  Spirocheta  pallida,  found  in  syphilis 
in  man.  There  are  also  spirochete  causing  diseases  of  domestic  birds 
and  others,  which  have  been  found  in  mammals  other  than  man,  but 
which  are  probably  not  very  pathogenic. 

Spirochete  in  Man. — SPIROCHETA  OBERMEIERI. — The  first  patho- 
genic spirillum  was  discovered  by  Obermeier  in  1868  in  the  blood  of 
persons  suffering  from  relapsing  fever.  Though  the  role  of  bacteria 
in  the  production  of  infectious  diseases  was  not  yet  well  recognized 
at  this  early  time,  it  was,  nevertheless,  believed  that  this  organism  was 
the  cause  of  the  disease.  All  spirochete  known  at  present  have 
resisted  every  attempt  at  artificial  cultivation,  and  they  are  not  as  well 
known  as  most  other  pathogenic  bacteria.  It  is  now  generally  believed 
that  the  Spirillum  Obermeieri  is  transmitted  from  sick  to  healthy 
persons  through  the  bites  of  bedbugs.  According  to  Novy  and 
Knapp  the  spirochete  of  European  and  Indian  relapsing  fever  are  not 
identical,  but  different  species. 

SPIROCHETA  DUTTONI. — Tick  fever,  a  disease  of  man  prevalent  in 
Equatorial  Africa,  was  shown  to  be  an  infection  due  to  spirochete. 
The  first  observations  concerning  its  nature  were  made  by  Ross  and 
Milne  in  Uganda  and  by  Button  and  Todd  in  Eastern  Congo.  This 
disease  is  transmitted  through  the  tick  Ornithodoros  moubata.  Robert 
Koch,  who  studied  the  disease  in  German  East  Africa,  demonstrated 
the  presence  of  spirochete  in  the  eggs  as  well  as  in  the  adult  ticks. 
Novy  has  shown  that  the  spirochete  of  African  tick  fever  is  a  species 
distinct  from  the  organism  of  European  and  Indian  relapsing  fever, 
and  he  has  proposed  the  name  of  Spirocheta  Duttoni  in  honor  of 
Dutton,  who  lost  his  life  while  studying  the  disease  in  Africa. 

SPIROCHETA  PALLIDA. — The  interest  in  the  study  of  spirochete 
was  enormously  increased  and  undertaken  by  hundreds  of  investi- 
gators when  Schaudin  and  Hoffmann  reported  that  they  had  found  an 
exceedingly  fine,  slender,  long,  and  very  typical  spirocheta  in  the 
primary  and  secondary  lesions  of  human  syphilis.  It  was  first  named 
Spirocheta  pallida,  later  Spironema  pallida,  and  still  later  Treponema 
pallidum,  but  it  is  now  most  commonly  known  in  literature  as 
Spirocheta  pallida,  or  the  spirocheta  of  syphilis. 

Spirocheta  pallida  is  found  in  almost  every  primary  syphilitic 
lesion;  often  in  secondary  but  very  rarely  in  tertiary  lesions.  It  is 
a  very  slender  spiral,  from  4  to  14  micra  long.  It  can  best  be  seen  in 
a  living  state,  and  unstained  by  the  aid  of  the  dark  field  illuminator. 


388         SPIRILLA,  PATHOGENIC  VIBRIONES,  SPIROCHETE 

It  shows  from  six  to  fourteen  windings  or  twists,  and  has  generally  a 
very  regular  corkscrew  shape.    It  is  very  lively  motile  in  a  manner 


FIG.  156 


FIG.  157 


Spirocheta  Obermeieri  blood  smear. 
Fuchsin.  X  1000.  (From  Itzerott  and 
Niemann.) 


Spirocheta  pallida  in  the  centre,  several 
bacilli  in  the  field.  India-ink  method.  X  1000. 
(Author's  preparation.) 


already  described  as  common  to  all  spirochete.  It  cannot  be  stained 
by  the  ordinary  staining  methods,  but  can  be  exhibited  by  the  Giemsa 
stain.  It  is  necessary  to  make  a  very  thin  smear  and  to  fix  the  air-dry 


FIG.  158 


FIG.  159 


Spirocheta  pallida,  part  of  one  which  shows 
the  typical  spiral  shape  and  unstained  spaces 
in  the  protoplasm.  Smear  from  primary  syphil- 
itic sore  stained  with  Goldhorn's  stain. 
X  1000.  (Author's  preparation.) 


Spirocheta  pallida  in  liver  of  a  case  of 
congenital  syphilis.  Levaditi's  silver  im- 
pregnation method.  X  1000.  (Author's 
preparation.) 


specimen  for  at  least  fifteen  minutes  in  absolute  alcohol.    The  ready- 
made  concentrated  Giemsa  stain  is  diluted  by  adding  one  drop  of  the 


SPIROCHETE  389 

stain  to  each  cubic  centimeter  of  distilled  water.  It  is  well  to  add  a 
few  drops  of  a  one-tenth  per  cent,  solution  of  carbonate  of  sodium 
and  a  few  drops  of  glycerin  to  about  30  c.c.  of  the  dilute  solution. 
The  stain  must  act  for  three  or  more  hours,  and  the  specimen  is  then 
washed  in  water  and  dried.  If  a  precipitate  has  formed  the  slide 
or  cover-glass  should  be  washed  rapidly  in  90  per  cent,  alcohol  and 
then  again  stained  for  some  time  with  the  dilute  Giemsa  stain  without 
the  addition  of  an  alkali.  The  best  method  to  show  the  Spirocheta 
pallida  rapidly  is  the  India-ink  method,  described  in  the  chapter  on 
Staining  Technique.  Spirilla  found  on  the  skin,  mucous  membranes, 
and  in  secretions  of  man  which  might  be  confounded  with  the  Spiro- 
cheta pallida  are  the  following:  Spirocheta  refringens,  Spirocheta 
balanitidis,  Spirocheta  buccalis,  Spirocheta  dentium,  and  Spirocheta 
pseudopallida  or  gracilis.  The  first  three  are  generally  much  coarser 
than  the  pallida,  and  stain  blue  with  Giemsa  stain,  while  the  pallida 
stains  pink.  The  last  two,  however,  are  almost  as  fine  as  the  pallida, 
and  as  they  also  stain  pink,  they  may  be  easily  mistaken  for  it. 

The  question  whether  the  Spirocheta  pallida  is  really  the  cause  of 
human  syphilis  is  not  fully  settled:  certain  discrepancies  still  remain 
to  be  cleared  up.  The  organism,  however,  is  generally  found  in 
primary  and  secondary  syphilitic  lesions,  and  it  is  of  great  diagnostic 
value  in  recognizing  the  disease. 

Spirochete  in  Birds. — Following  the  discovery  of  the  Spirillum 
Obermeieri,  certain  spirochete  pathogenic  for  birds  were  found, 
and  later  others  in  mammals  and  man.  They  were  found  in  diseases 
of  domestic  birds,  and  resembled  the  Spirocheta  Obermeieri,  which 
causes  recurrent  fever  in  man.  The  first  organism  of  this  type  was 
found  in  geese  in  1893  by  Sakharoff  in  Russia,  and  named  accordingly 
Spirocheta  anserina.  The  same  bacterium  was  seen  by  Ducleaux  in 
Tunis,  likewise  in  geese.  Marchoux  and  Salimbeni  (1903)  found 
spirochete  in  Brazil  in  chickens,  and  named  them  Spirocheta  galli- 
narum.  Since  then  spirochete  have  been  reported  from  Rhodesia, 
India,  Soudan,  Algiers,  Tunis,  Cyprus,  Martinique,  Bulgaria,  and  the 
last  report  by  Dodd  comes  from  Queensland.  Spirillosis  of  domestic 
birds,  therefore,  appears  to  occur  over  a  widespread  area.  The 
disease  causes  fever,  diarrhea,  and  emaciation,  and  either  ends  fatally 
within  a  few  days  or  leads  to  recovery.  Postmortem  examination  of 
dead  animals  shows  enlargement  of  the  spleen,  enlargement  and  fatty 
degeneration  of  the  liver,  and  sometimes  fatty  degeneration  of  the 
myocardium,  with  fibrinous  deposits  on  the  endocardium.  The 
spirilla  are  found  in  the  blood  during  the  disease.  They  disappear, 
however,  before  death,  and  cannot  be  found  in  the  cadaver;  they  also 
disappear  in  the  case  of  recovery.  A  single  attack  protects  against 
subsequent  infection.  The  disease  is  conveyed  from  one  animal  to 
another  through  parasitic  fowl  ticks  (Argas  miniatus),  in  the  body 
of  which  the  organism  can  evidently  remain  alive  for^a  long  time. 
The  disease  can  also  be  transferred  by  artificial  inoculation  of  the 


390         SPIRILLA,  PATHOGENIC  VIBRIONKS,  SPIROCHETE 

blood  of  infected  birds  into  healthy  ones.  Young  birds  generally  die 
after  an  artificial  infection.  An  immune  serum  may  also  be  prepared 
by  repeated  injections  of  blood  containing  spirilla  into  a  horse. 

Spirochete  in  Mammals. — Organisms  of  this  type  have  been  found  by 
Theiler  in  the  Transvaal  in  the  blood  of  cattle  simultaneously  infected 
with  Piroplasmata  and  Trypanosomata.  The  spirilla  seen  were 
slender  and  20  to  30  micra  long,  and  very  similar  to  those  described 
in  birds.  Laveran  named  this  organism  Spirillum  Theileri.  It  is  not 
known  whether  it  is  pathogenic  or  not.  The  observations  of  Theiler 
on  cattle  have  been  confirmed  by  Ziemann  in  the  Cameroon  and  by 
Robert  Koch  in  East  Africa.  These  spirochete  of  cattle  are  transmitted 
through  the  bite  of  the  tick  Rhipicephalus  decoloratus,  which  may  at 
the  same  time  transmit  Texas  fever,  as  shown  by  certain  experiments 
of  Laveran  and  Vallee.  Theiler  also  found  spirochete  in  sheep  in 
the  Transvaal,  and  he  and  other  authors  have  a  few  times  found  the 
organisms  in  horses.  Spirochete  have  also  been  seen  in  rats  in  India, 
and  Nicolle  and  Comte  found  them  in  a  common  bat  (Vespertilio 
Kuhli)  in  Tunis.  The  latter  spirilla  are  12  to  18  micra  long,  not  more 
than  one-quarter  of  a  micron  thick.  They  have  pointed  ends  and 
divide  by  binary  division  at  right  angles  to  the  long  axis.  The  infection 
can  be  transmitted  from  sick  to  healthy  animals. 

FIG.  160 


Spirocheta  pallida,  twisted  and  intertwined  form,  primary  lesion  of  syphilis.     Goldhorn's 
stain.      X  1000.     (Author's  preparation.) 

Are  Spirochete  Bacteria  or  Protozoa? — Extensive  studies  on  the 
spirochete  of  relapsing  and  tick  fevers  and  other  organisms  of  this 
group  have  led  Novy  and  Knapp  to  agree  with  the  conclusions 
previously  drawn  by  Carter,  Norris,  Martin,  Borrel,  R.  Koch,  and 
others  that  spirochete  are  bacteria,  i.  e.,  vegetable  microorganisms, 
and  not  Protozoa,  as  Schaudin  and  other  investigators  have  believed. 
In  the  examination  of  living  spirochete,  Novy  and  Knapp  failed  to 
observe  the  presence  of  an  undulating  membrane  with  a  flagellum 


QUESTIONS  391 

as  encountered  in  trypanosomes.1  They  found  the  contents  of  the 
spirochetal  body  perfectly  homogeneous,  without  any  indication  of  a 
nucleus  and  a  blepharoblast.  The  flagella  had  the  characteristics 
of  bacterial  organs  of  locomotion  and  not  those  found  in  protozoa. 
On  the  Spirocheta  Obermeieri  they  were  able  to  demonstrate  a  slender 
whip  as  long  as  the  body  of  the  organism,  wavy,  like  the  flagella  of 
other  bacteria,  and  not  coarse  and  thick  like  the  flagella  of  trypan- 
osomes. They  observed  evidences  of  transverse  binary  division  at 
right  angles  to  the  long  axis  both  in  living  and  stained  preparations. 
They  also  ascertained  that  spirochete  included  in  very  thin  collodion 
sacs  and  exposed  to  the  action  of  running  distilled  water  or  directly 
mixed  with  distilled  water  showed  plasmolytic  changes  like  bacteria 
and  not  like  the  much  more  susceptible  and  delicate  protozoa,  par- 
ticularly trypanosomes.  They  further  demonstrated  that  spirochete 
.are  not  as  susceptible  to  higher  temperatures  as  trypanosomes,  but 
act  more  like  bacteria,  and  do  not,  under  elevated  temperatures,  change 
their  shape  and  form  like  trypanosomes.  In  their  rapid  method  of 
multiplication  when  injected  into  susceptible  animals  and  in  the 
production  of  immunity  spirochete  likewise  act  like  bacteria  and  not 
like  trypanosomes.  The  author  has  had  opportunity  to  study  Spiro- 
cheta pallida  extensively  both  in  the  live  state  with  the  dark  field 
illuminator  and  in  stained  and  silvered  preparations,  and  he  likewise 
believes  that  spirochete  are  bacterial  and  not  protozoan  organisms, 
that  they  do  not  possess  an  undulating  membrane,  a  nucleus,  or  a 
blepharoblast  like  the  protozoan  trypanosomes,  and  that  they  divide 
at  right  angles  to  the  long  axis  like  other  bacteria.  Anyone  who  has 
studied  a  large  number  of  stained  specimens  of  Spirocheta  pallida 
must  have  repeatedly  seen  forms  where  the  division  in  the  middle 
of  the  long  axis  was  clearly  indicated  and  almost  complete.  The 
forms  mistaken  for  spirocheta  dividing  by  splitting  parallel  with 
the  long  axis  are  simply  a  pair  of  intertwined  spirals  and  not  a  dividing 
organism. 

QUESTIONS. 

1.  Name  and  give  the  characteristics  of  the  two  groups  of  spiral  bacteria. 

2.  Describe  the  type  of  motility  of  vibrio  and  of  spirocheta. 

3.  Where  was  the  vibrio  of  Metchnikoff  first  found?    What  disease  does  it 
produce  ? 

4.  Describe   the   disease   caused   in   chickens  by   the  Vibrio  Metchnikovi. 
Describe  the  pathologic  lesions. 

5.  What  names  have  been  given  to  this  disease  in  chickens? 

6.  Describe  the  morphology  of  the  Vibrio  Metchnikovi. 

7.  Describe  its  cultural  properties. 

8.  Does  it  always  liquefy  gelatin? 

9.  What  is  the  difference  in  animal  inoculations  of  the  Vibrio  Metchnikovi 
and  of  the  vibrio  of  Asiatic  cholera? 

10.  What  animal  diseases  are  caused  by  the  vibrio  of  Asiatic  cholera? 

1  The  student  should  compare  the  description  as  given  in  the  chapter  on  Trypanosomes, 
with  the  facts  here  given  as  to  the  morphology  of  spirochete. 


392        SPIRILLA,  PATHOGENIC  VIBRIONES,  SPIROCHETE 

11.  What  are  the  characteristic  cultural  differences  between  the  vibriones 
of  Asiatic  cholera  and  of  Metchnikoff? 

12.  What  is  the  test  for  the  presence  of  nitrites  and  indol  in  culture  media? 

13.  Name  and  describe  some  other  non-pathogenic  vibriones. 

14.  What  was  the  first  pathogenic  spirocheta  discovered?    What  disease  does 
it  produce?    How  is  this  disease  spread? 

15.  What  is  the  cause  of  .African  tick  fever.    How  is  it  spread? 

16.  Describe  the  morphology,  staining,  and  cultural  properties  of  Spirocheta 
pallida. 

17.  In  what  disease  is  it  found?    How  can  its  presence  best  be  demonstrated? 

18.  Describe  some  diseases  caused  by  spirocheta  in  domestic  birds.    How  are 
those  diseases  spread? 

19.  State  in  what  mammals  spirochete  have  been  found  and  what  is  known 
about  their  pathogenesis. 

20.  Discuss  the  question  whether  spirochete  are  protozoa  and  near  relatives 
of  trypanosomes  or  not. 


CHAPTER    XXXIV. 

THE  BACILLUS  LACTIMORBI  OF  TREMBLES. 

Occurrence. — The  disease  known  as  milk  sickness,  sick  stomach, 
swamp  sickness,  tires,  trembles,  slows,  etc.,  is  an  affection  of  cattle, 
and  occasionally  of  sheep.  It  has  been  observed  in  the  United  States 
for  over  one  hundred  years,  and  apparently  has  never  been  described 
in  any  other  part  of  the  world.  It  was  formerly  more  frequently 
mentioned,  and  has  evidently  decreased  considerably.  An  affected 
animal  is  lifeless,  tired  out  on  the  slightest  exertion,  and  the  muscular 
weakness  is  manifested  by  trembling,  which  characteristic  symptom 
has  led  to  the  designation  "trembles."  In  a  more  advanced  stage 
there  is  stiffness  of  the  joints,  great  weakness  and  the  animal  is  unable 
to  get  up  after  it  has  once  fallen  to  the  ground. 

Pathologic  Lesions. — Jordan  and  Harris  describe  the  pathologic 
lesions  of  the  disease  in  cattle  as  follows :  There  are  no  characteristic 
external  changes,  but  upon  opening  the  body  the  smell  of  acetone 
can  often  be  detected.  Occasionally  a  small  quantity  of  clear  yellow 
fluid  is  present  in  the  pleural  cavity.  The  lungs  are  edematous.  The 
visceral  layer  of  the  pericardium,  chiefly  along  the  course  of  the  cardiac 
veins  from  base  to  apex,  and  around  the  roots  of  the  large  vessels, 
shows  ecchymotic  spots,  which  are  also  occasionally  seen  in  the 
parietal  layer  of  the  peritoneum.  The  liver  is  much  enlarged,  of  a 
purple  red  color,  and  much  congested;  it  appears  mottled  in  con- 
sequence of  the  presence  of  areas  of  fatty  degeneration,  of  which  it 
shows  marked  evidences  on  section;  the  consistency  of  the  organ  is 
much  diminished.  The  spleen  is  not  enlarged.  The  kidneys  are 
enlarged,  congested,  and  sometimes  show  evidences  of  parenchymatous 
degeneration.  The  mucosa  of  the  small  intestines  is  congested,  and 
shows  ecchymotic  spots,  and  the  jejunum  and  the  upper  part  of  the 
ileum  are  covered  by  a  yellowish,  very  tenacious  mucus. 

The  disease  appears  to  be  usually  contracted  by  grazing  cattle  or 
sheep  which  have  entered  an  infected  territory.  It  may  then  be 
communicated  to  man  through  raw  milk  or  butter,  or  through  raw 
or  insufficiently  cooked  meat;  dogs  and  cats  may  also  become  infected. 
The  mortality  in  man  is  claimed  to  be  high,  but  this  is  probably 
incorrect,  because,  as  a  rule,  severe  cases  only  are  recognized  while  the 
milder  cases  have  escaped  notice. 

Morphology  and  Staining  Properties. — Jordan  and  Harris  isolated 
an  aerobic  organism  which  they  called  Bacillus  lactimorbi  from  the 
internal  organs  of  cattle  dead  from  trembles.  They  describe  it  as 
follows:  In  cover-glass  preparations  made  from  the  juice  of  the 


394  THE  BACILLUS  LACTIMORBI  OF  TREMBLES 

organs  the  bacilli  are  longer  and  more  slender  than  the  colon  bacillus; 
they  stain  occasionally  unevenly  with  methylene  blue.  In  preparations 
made  from  cultures  grown  on  agar  at  37°  C.,  the  organism  is  found 
to  be  a  rod,  a  little  smaller  than  the  anthrax  bacillus,  occurring  singly 
and  in  pairs  and  in  occasional  filaments.  As  a  rule,  the  rods,  at  the 
end  of  twenty-four  hours'  incubation,  do  not  stain  deeply  with 
methylene  blue,  even  if  the  solution  be  slightly  heated,  but  at  one  or 
both  poles  and  at  the  centre  of  each  rod  metachromatic  granules  are 
found  which  take  on  a  reddish  or  purple  tint.  In  young  cultures  the 
bacilli  are  Gram  positive.  Spore  formation  occurs  after  twenty-four 
hours'  incubation,  the  spores  may  be  first  oval,  but  when  completely 
mature  they  are  round.  They  lie  near  one  end  of  the  rod.  The 
organism  is  motile  and  possesses  ten  to  fifteen  peritrichous  flagella, 
which  can  be  demonstrated  by  van  Ermengem's  method. 

Cultural  Properties. — The  cultural  characteristics  of  the  organism 
are  as  follows:  On  agar  slants  incubated  at  37°  C.,  at  the  end  of 
twenty-four  hours,  the  surface  is  more  or  less  irregularly  covered 
by  a  delicate  veil-like  growth,  which  is  more  profuse  at  the  end  of 
from  forty-eight  to  seventy-two  hours;  on  some  cultures  it  may 
eventually  take  on  a  semiviscid  character.  The  color  is  grayish, 
moist,  smooth,  and  glossy;  there  is  no  pigmentation  of  the  growth 
itself  or  of  the  medium.  There  is  no  gas,  the  condensation  water- 
growth  is  heavy,  gray  white  in  color;  no  odor  being  present.  No 
gas  is  formed  in  glucose  agar  cultures.  In  bouillon,  at  the  end  of 
twenty-four  hours,  no  growth  except  sometimes  a  slight  clouding  at 
the  surface,  may  be  noticeable.  At  the  end  of  twenty-four  hours  a 
well-formed  pellicle,  which  will  sink  if  the  tube  is  agitated,  appears  on 
the  surface.  The  remainder  of  the  medium  is  feebly  clouded,  and 
there  may  be  a  fine  semiflocculent  sedimented  growth  at  the  bottom 
of  the  tube.  Milk  becomes  alkaline  after  a  number  of  days,  but 
coagulation  does  not  occur;  there  is  no  growth  on  potatoes.  In  gelatin 
stab  cultures,  kept  at  20°  C.,  the  growth  begins  to  appear  at  the  end 
of  forty-eight  hours,  but  is  not  well  developed  until  at  the  end  of 
seventy-two  hours.  There  is  little  or  no  surface  growth,  but  down 
the  stab  to  the  extent  of  about  2  centimeters,  a  delicate,  smooth, 
gray  growth  appears.  At  the  end  of  a  week  the  surface  growth  has 
extended  as  a  fairly  well-developed  film  of  a  whitish  color,  smooth, 
moist,  and  glossy;  at  times  the  first  evidence  of  liquefaction  may  now 
be  detected,  but  this  is  not  well  established  until  the  tenth  day,  when 
the  medium  is  slightly  fluid  just  beneath  the  surface  growth.  As  time 
passes  liquefaction  slowly  progresses.  The  growth  in  gelatin  leads  to 
a  more  or  less  well-defined  putrescent  odor.  Agar  plate  colonies  at  the 
end  of  twenty-four  hours'  incubation  at  30°  to  37°  C.  resemble  those 
of  the  Streptococcus  pyogenes,  but  with  this  difference,  that  they  show 
a  tendency  to  spread  out  in  a  film,  particularly  if  the  agar  is  freshly 
made;  in  the  latter  case  the  whole  surface  of  the  plate  may  be  covered. 
In  gelatin  plates  grown  at  20°  C.  the  colonies  make  their  appearance 
at  the  end  of  from  forty-three  to  seventy-two  hours,  and  resemble  at 


QUESTIONS  395 

first  those  of  streptococci,  but  at  the  end  of  the  fourth  day  they  appear 
more  vigorous,  and  the  surface  colonies  partake  more  of  the  character- 
istics of  Staphylococcus  albus,  although  the  color  is  of  a  yellowish- 
white  character. 

The  thermal  death  point  of  the  organism  was  ascertained  to  be 
five  minutes'  exposure  to  55°  C.  for  the  vegetative  form,  fifteen 
minutes  at  100°  C.  for  the  spores. 

Agglutination  tests  of  the  serum  of  cattle  and  man  with  the  Bacillus 
lactimorbi  did  not  furnish  any  uniform  results.  Some  sera  would 
agglutinate  some  stems  of  bacilli  in  dilution  of  1  to  50  or  200;  others 
would  not  be  agglutinated,  and  some  stems  of  the  organism  could 
not  be  agglutinated  by  any  sera. 

Jordan  and  Harris  found  the  Bacillus  lactimorbi,  or  an  organism 
not  distinguishable  from  it  by  any  of  the  tests  applied,  in  the  soil  of 
regions  where  milk  sickness  has  never  been  known;  they  found  it  in 
normal  cow  dung,  on  various  grain  and  forage  plants. 

Luckhardt  isolated  an  identical  organism  from  dried  alfalfa  from 
Wisconsin,  from  the  same  material  from  Illinois  and  Indiana,  and 
from  four  weeds  collected  by  Jordan  and  Harris  in  the  Pecos  Valley 
in  New  Mexico,  where  they  first  studied  the  disease.  Of  six  dogs 
inoculated  with  cultures  of  these  stems  of  bacilli  obtained  by  Luck- 
hardt, two  showed  in  a  slight  degree  the  symptoms  observed  in  milk 
sickness.  Luckhardt  concludes  from  his  work  with  the  Bacillus 
lactimorbi  that  it  is  remarkable  that  if  this  bacillus  be  the  cause  of 
milk  sickness  it  should  have  so  wide  a  distribution  in  milk-sick  and 
non-milk-sick  regions.  It  is  also  apparent  that  loss  of  virulence 
occurs  after  a  time  in  the  races  of  the  organisms  isolated,  and  that  no 
feeding  experiments  have  so  far  yielded  uniformly  any  well-defined 
pathologic  picture  of  the  disease. 

The  animal  experiments  of  Jordan  and  Harris  have  likewise 
lacked  uniformity  and  definiteness  in  their  outcome.  On  the  whole, 
it  cannot  yet  be  considered  as  established  that  the  Bacillus  lactimorbi 
is  indeed  the  cause  of  trembles.  Some  of  the  results  of  the  animal 
experiments  made  with  the  blood  of  dead  animals  and  with  pure 
cultures  of  the  bacillus  rather  point  to  the  possibility  of  an  ultra- 
microscopic  virus. 

QUESTIONS. 

1.  What  other  names  have  been  given  to  milk  sickness  in  cattle? 

2.  Where  has  the  disease  been  encountered? 

3.  What  are  its  most  characteristic  symptoms? 

4.  Describe  the  pathologic  changes  found  after  death  from  this  disease. 

5.  Is  the  disease  communicable  to  man,  and  how? 

6.  Describe  the  morphology  and  staining  properties  of  the  Bacillus  lactimorbi. 

7.  Describe  a  culture  on  agar. 

8.  How  does  the  organism  affect  milk  when  grown  in  it? 

9.  Describe  its  growth  in  gelatin. 

10.  What  is  the  thermal  death  point  of  the  vegetative  form  and  of  the  spores 
of  the  organism? 

11.  What  has  been  the  result  of  agglutination  tests  with  the  organism? 

12.  Where  has  the  Bacillus  lactimorbi  been  found  outside  of  the  bodies  of 
animals  dead  from  trembles? 


CHAPTER    XXJXV. 

A  LOCAL  EQUINE  DISEASE  AND  A  BACILLUS  OF  THE  SUBTILIS 

GROUP. 

IN  the  neighborhood  of  Lake  Winnebago  and  in  other  places  in 
Wisconsin  a  disease  among  horses  characterized  by  a  rather  rapid 
onset  has  been  frequently  observed  in  the  late  summer  or  early  fall. 
The  animal  appears  to  be  perfectly  well  in  the  evening,  the  following 
morning  it  refuses  to  eat  or  drink,  inclines  the  head  downward, 
breathes  very  rapidly,  has  a  rapid  pulse  of  about  80,  and  a  temperature 
ranging  from  104°  to  107°  F.  The  stools  are  first  loose,  and  a  profuse 
diarrhea,  which  reaches  its  maximum  in  about  three  days,  develops 
rapidly.  Sometimes  the  bowels  are  not  loose  but  rather  constipated, 
and  then  pulmonary  symptoms  develop.  The  animals  so  affected 
generally  die. 

The  author,  in  conjunction  with  Dr.  O.  N.  Johnson,  of  Appleton, 
Wis.,  in  August,  1908,  examined  bacteriologically  a  number  of  cases  of 
this  affection  occurring  in  Dr.  Johnson's  practice.  Blood  was  obtained 
from  the  jugular  vein  of  sick  animals  under  aseptic  precautions  in 
8  cases  and  immediately  distributed  to  culture  tubes  and  flasks.  In  5 
cases  an  identical  organism  was  obtained.  It  was  a  rather  large,  lively 
motile,  Gram-positive  bacillus,  which  grew  best  at  room  temperatures, 
less  rapidly  at  the  incubator  temperature  (37°  C.).  On  agar  it 
formed  a  white,  moist,  shining,  rather  rapidly  spreading  growth; 
gelatin  in  stick  cultures  was  rapidly  liquefied,  with  the  formation  of  a 
funnel-shaped  zone  of  liquefaction;  it  produced  acid  in  litmus  milk 
and  coagulated  it  after  seventy-two  hours;  on  glucose  agar  it  did  not 
grow  as  well  as  in  plain  agar  along  the  stab,  but  grew  well  on  the 
surface  and  did  not  produce  any  gas.  In  bouillon  turbidity  it  was 
produced  rapidly,  but  no  pellicle  was  formed  on  the  surface.  Spore 
formation  occurred  in  various  media  after  twenty-four  hours'  incu- 
bation at  37°  C.  One  of  the  stems  examined  had  not  coagulated  milk 
after  seventy-two  hours  and  another  stem  formed  a  dry,  hard  pellicle 
on  bouillon.  In  other  respects  the  action  of  the  five  stems  was  identical. 
On  the  whole,  the  organism  appeared  to  be  a  member  of  the  subtilis 
group,  but  it  seems  hardly  possible  that  it  was  an  outside  contami- 
nation, as  it  developed  in  the  blood  inoculations  from  five  out  of  eight 
horses  examined. 

Emulsions  of  the  organism  raised  on  agar  slants  and  prepared 
with  physiologic  salt  solution  were  injected  intraperitoneally  into  a 
number  of  guinea-pigs.  During  the  first  three  or  four  days  no  visible 


A  LOCAL  EQUINE  DISEASE  397 

effect  was  produced  in  the  inoculated  animals.  On  the  morning  of  the 
fifth  day  guinea-pigs  inoculated  from  cultures  Nos.  1,  2,  and  3  (each 
from  a  different  horse)  were  found  dead.  On  the  seventh  day  the 
guinea-pig  inoculated  from  stem  No.  5  was  found  dead.  Autopsies 
on  the  dead  animal  showed  no  typical  lesions.  Cultures  were  made 
from  the  heart's  blood  of  the  four  dead  guinea-pigs;  in  3  cases  an 
organism  identical  with  the  one  injected  was  obtained  but  the  cultures 
soon  died  out.  One  which  remained  alive  after  several  transplants 
was  inoculated  subcutaneously  into  a  healthy  horse.  It  failed  to 
produce  any  general  reaction,  the  temperature  of  the  horse  was  taken 
regularly,  and  remained  normal  for  a  number  of  days.  A  slight  local 
reaction  developed  at  the  site  of  the  injection  (neck),  but  this  dis- 
appeared after  a  few  days.  In  the  bacteriologic  part  of  this  investiga- 
tion the  author  was  assisted  by  Dr.  Conrad  Jacobson.  No  definite 
conclusion  as  to  the  relation  of  the  bacillus  obtained  to  the  disease  can 
be  drawn  from  the  above  results.  Further  and  more  extensive  studies 
are  necessary. 


CHAPTER    XXXVI. 

LOWER  HYPHOMYCETES— TRICHOMYCETES—LEPTOTHRIX— 
CLADOTHRIX— STREPTOTHRIX  AND  ACTINOMYCES. 

HYPHOMYCETES. 

THE  organisms  discussed  in  the  preceding  pages  have  been  con- 
sidered chiefly  from  the  point  of  view  of  being  the  cause  or  etiologic 
factor  of  disease  in  domestic  animals  and  man.  All  of  them  are 
exceedingly  simple,  unicellular,  vegetable  microbes,  and  they  are 
known  as  bacteria  or  schizomycetes.  The  term  schizomycetes  denotes 
fission  fungi  and  is  descriptive  of  the  characteristic  mode  of  binary 
division  or  fission  common  to  all  organisms  belonging  to  the  bacteria. 
True  branching1  does  not  occur  among  the  latter,  while  the  higher 
fungi,  eumycetes  or  hyphomycetes,  do  form  genuine  branches.  For 
this  reason  they  may  be  defined  as  vegetable  microorganisms  composed 
of  elongated  or  filiform  cells,  which  form  genuine  branches  and  which 
multiply  by  special  organs  or  cells  called  spores.  Their  development 
is  characterized  by  the  formation  of  true  branches  from  the  original 
cell  or  main  trunk,  and  the  latter  and  the  branches  form  one  con- 
tinuous mass  of  protoplasm.  The  branching  may  be  a  comparatively 
simple  arrangement  as  in  the  lower  eumycetes,  or  it  may  be  relatively 
complicated  with  a  vast  network  of  dichotomous  and  pseudodicho- 
tomous  divisions  as  in  the  higher  eumycetes.  The  term  true  dicho- 
tomous division  designates  a  division  into  two  equal  branches,  while 
pseudodichotomous  division  indicates  that  one  branch,  which  is 
continued  as  the  principal  stem,  sends  out  a  lateral,  often  smaller 
branch. 

When  the  hyphomycetes  or,  as  they  are  commonly  called,  the 
moulds  are  studied  a  differentiation  of  the  organism  into  two  parts 
which  perform  different  physiologic  functions  can  be  distinguished. 
One  part,  the  mycelium,  presents  itself  as  a  more  or  less  branched 
mass  which  serves  for  the  nutrition  and  preservation  of  the  individual 
organism,  while  the  second  part,  known  as  the  fructification  organ, 
produces  the  spores  and  serves  for  the  preservation  of  the  species. 
The  entire  soft  cellular  structure,  without  any  wooden  fibers,  of  which 
the  whole  body  of  the  mould  consists,  is  known  as  the  thallus. 

1  Bacteria  like  the  tubercle  bacillus  are  not  taken  into  consideration  at  this  place,  notwith- 
standing the  fact  that  this  organism  sometimes  forms  true  branches,  indicating,  perhaps, 
that  it  should  be  classified  among  the  lower  eumycetes  rather  than  among  the  bacteria.  Since 
such  organisms  have  always  been  classified  among  the  bacteria  in  medical  considerations,  it 
is  not  desirable  to  make  any  change. 


SPORE  FORMATION  399 

According  to  Lafar,  the  mycelium  may,  therefore,  be  defined  as 
that  portion  of  the  thallus  of  the  mould  which  is  spread  out  on  the 
culture  soil  and  which  derives  from  it  the  nutrition  necessary  for  the 
organism.  The  mycelium  arises  from  a  spore  which  has  been  im- 
planted upon  or  within  a  favorable  culture  soil.  From  the  spore  one 
or  several  germ  sacs  are  formed ;  these  grow  elongate,  divide,  and  form 
the  individual  hyphen  or  mould  filaments  which  in  their  entity  compose 
the  mould  mycelium.  The  latter  always  grows  at  the  external  ends 
of  the  individual  hyphens  and  not  at  the  end  which  originally  arose 
from  the  spore.  In  this  respect  the  eumycetes  differ  from  the  schizo- 
mycetes,  which  grow  at  both  ends  and  in  multiplication  divide  in  the 
middle.  In  the  study  of  the  mycelium  two  types  are  noted,  one  in 
which  the  whole  mass,  much  branched  and  complicated  as  it  may 
be,  consists  of  a  single  cell  only;  the  other  in  which  the  hyphens,  or 
filaments,  are  divided  by  septa  into  cylindrical  segments,  which  by 
their  union  form  the  hyphens.  Moulds  of  the  first  type  are  classified 
as  phycomycetes;  those  of  the  latter  as  mycomycetes.  The  fructifying 
organ  is  also  developed  in  all  hyphomycetes,  though  in  the  lowest 
forms  it  may  be  rather  rudimentary  and  inconspicuous.  The  function 
of  the  fructifying  organ  is  the  production  of  cells  from  which  new 
individuals  can  be  formed.  Such  cells  of  lower  plants,  as  has  already 
been  stated  in  the  consideration  of  bacteria,  are  called  spores.  A 
bacterium,  however,  almost  invariably  forms  a  single  spore  only, 
while  hyphomycetes  generally  form  a  large  number  of  spores. 

Spore  Formation. — Four  different  varieties  of  spore  formation  are 
recognized  in  hyphomycetes,  namely: 

1.  Endospores,  or  gonidia. 

2.  Zygospores 

3    Exospores,  or  conidia. 

4.  Chlamydospores,  or  gemmae. 

The  first  three  types  of  spores  are  formed  by  the  fructifying  organ, 
except  among  the  very  lowest  type  of  hyphomycetes,  where  the  cells 
of  the  mycelium  form  the  spores  in  their  interior  without  any  pre- 
liminary differentiation.  This  mode  of  formation  is  like  that  in 
bacteria,  only  that  it  is  generally  a  multiple  and  not  a  single  spore 
formation. 

1.  ENDOSPORES,  OR  GONIDIA. — In  the  formation  of  the  fructifying 
organ  one  or  several  hyphens  arise  from  the  mycelium  and  grow 
upward,  becoming  straight  and  much  stronger  than  the  ordinary 
mycelial  hyphens  or  filaments.  This  stem  is  called  the  fruit  bearer, 
or  fruit  carrier.  Its  upper  free  end  becomes  globularly  or  elliptically 
enlarged,  and  in  this  extremity  the  spores  are  formed  after  it  has 
become  separated  from  the  remainder  of  the  stem  by  a  straight  or 
curved  septum.  This  free  round  or  oval  separate  end  forming  the 
spores  is  now  called  a  sporangium,  while  the  stem  which  carries 
the  latter  is  often  called  the  columella  (the  little  pillar).  This  is  the 
mode  in  which  endospores  or  gonidia  are  generally  formed. 


400  HYPHOMYCETES 

2.  ZYGOSPORES. — This  variety  of  spores   is  formed   as   follows: 
The  outer,  free  ends  of  two  hyphens,  or  filaments,  swell  up  and 
become  club-shaped.    The  clubs  touch  each  other  and  the  membranes 
separating  them  become  fused.     The  united  clubs  at  the  same  time 
become  divided  from  the  rest  of  their  hyphens  by  two  septa.    In  this 
way  two  separate  cells  touching  each  other  are  formed.     These  are 
called  copulation  cells,  or  gametes,  while  the  upper  part  of  the  hyphens 
which  carry  the  latter  are  known  as  the  carrying  cells,  or  suspensors. 
The  partition  wall,  or  septum,  between  the  two  gametes  touching  each 
other  finally  become  dissolved,  and  from  the  contents  of  the  two 
copulation  cells  the  spore,  known  as  a  zygospore,  is  formed.    Spores 
are  also  sometimes  formed  from  a  single  hyphen  without  the  union 
of  two  gametes,  and  this  variety  of  spores  is  called  azygospores,  or 
parthenospores. 

3.  EXOSPORES,  OR  CONIDIA. — These  may  be  formed  by  either  of 
the  following  ways:     In  some  hyphomycetes  the  fruit  bearer  at  its 
upper  extremity  forms  a  round  end  which  becomes  separated  from 
the  rest  of  the  filament  by  a  septum.      The  cut-off  segment  then 
assumes  a  round  or  oval  shape,  forming  thus  the  first  spore.     The 
end  of  the  hyphen  to  which  the  first  spore  adheres  repeats  the  process, 
and  a  second  spore  is  formed  situated  between  the  first  spore  and  the 
end  of  the  hyphen.    This  process  continues,  and  gradually  a  row  of 
spores,  of  which  the  outermost  is  the  oldest  and  the  innermost  the 
youngest,  becomes  attached  to  the  fruit-bearing  hyphen.    In  the  other 
method  of  spore  formation  the  first  spore  is  formed  in  the  manner 
just  described,  but  then  it  itself  divides,  and  this  process  continuing, 
a  row  of  spores  is  formed  in  which  the  outermost  is  the  youngest  and 
the  innermost  the  oldest.    The  first  mode  of  spore  formation  proceed- 
ing from  the  inner  end  of  the  row  is  called  basipetal  conidia  formation; 
the  second,  in  which  the  new  spores  are  formed  at  the  outer  end,  is 
known  as  basifugal  conidia  formation. 

CHLAMYDOSPORES,  OIDIA,  OR  GEMMAE  FORMATION. — In  this  form  of 
spore  formation  a  fruit  bearer  proper  is  not  formed,  but  the  filaments 
of  the  mycelium  break  up  into  small  segments.  Each  of  these  when 
falling  upon  a  fertile  soil  may  form  a  new  hyphomycete  individual. 
This  mode  of  spore  formation  was  first  observed  in  a  fungus  known 
as  Oidium  lactis,  and  for  this  reason  it  is  also  called  oidia  formation. 
The  small  segments  arising  in  gemmse  formation  often  assume  a  more 
marked  spore  character  by  surrounding  themselves  with  a  tough, 
tenacious,  protecting  membrane. 

Resistance  of  Spores. — The  resistance  of  spores  of  hyphomycetes 
is  generally  like  that  of  the  spores  of  bacteria,  and  like  them  they  are 
more  resistant  than  the  adult  form  of  the  organism.  As  a  rule,  however, 
their  resistance  is  not  as  great  as  that  of  bacteria,  though  some  may 
withstand  drying  out  for  many  years.  Hansen  has  shown  that  the 
spores  of  certain  common  moulds  survived  after  a  desiccation  of 
from  eight  to  twenty-one  years.  Pasteur  has  shown  that  the  conidia  of 


CLASSIFICATION  401 

the  common  blue  mould  (Penicillium  glaucum)  while  killed  by  boiling- 
water  can  withstand  dry  heat  at  120°  C.  for  some  time. 

Classification. — In  considering  now  some  of  the  pathogenic  micro- 
organisms of  a  higher  development  and  more  complicated  morphology 
than  the  bacteria,  the  classification  proposed  by  Lackner-Sandoval 
and  adopted  by  Petruschky  in  his  contribution  on  pathogenic  tricho- 
mycetes  in  Kolle  and  Wassermann's  Manual  will  be  followed.  Pet- 
ruschky divides  the  hyphomycetes  into  the  trichomycetes  (hair  fungi) 
and  the  higher  hyphomycetes.  The  former  are  again  subdivided 
into  (1)  leptothrix,  (2)  cladothrix,  (3)  streptothrix,  and  (4)  actino- 
myces. 

The  first  two  genera  are  nearly  related  to  the  bacteria  or  schizomy- 
cetes,  while  the  latter  two  are  clearly  members  of  the  order  of  hypho- 
mycetes or  eumycetet, 

1 .  LEPTOTHRIX. — These  present  themselves  as  stiff,  slender  filaments 
which  do  not  show  any  branching  and  on  which  dividing  processes 
can  rarely  be  observed. 

2.  CLADOTHRIX. — These    are    filaments    with    pseudobranching. 
The  branching  effect  is  produced  by  a  protrusion  of  the  protoplasm 
through  the  membrane  and  a  rapid  breaking  up  of  the  filaments  into 
short  rods  which  assu  me  the  character  of  bacilli. 

3.  STREPTOTHRIX. — This  genus  forms  a  true  branching  mycelium 
with  septa  formation  and  the  production  of  fruit-bearing  filaments. 
The  latter  break  up  into  short  segments  which  form  chains  of  exo- 
spores,  or  conidia. 

4.  ACTINOMYCES. — This  is  really  a  streptothrix  and  it  systematically 
does  not  differ  from  the  latter.     It  shows,  however,  the  peculiar 
property  when  invading  tissues  as  a  pathogenic  microorganism  of 
forming  in  the  pathologic  lesions  very  characteristic  stars,  composed 
of  the  ends  of  swollen  hyphons  or  filaments,  arranged  in  rosette  form. 
On  this  account  the  organism  has  received  the  name  of  ray  fungus, 
or  actinomyces,  and  it  is  well  to  retain  it  and  classify  separately  the 
pathogenic  streptothr.ces  showing  this  property. 

1.  Leptothrices. — The?.e  are  represented  by  a  species  frequently 
found  in  the  mouths  of  persons  and  known  as  Leptothrix  buccalis. 
The  organism  is,  as  a  rule,  a  harmless  saprophyte,  but  it  has  some- 
times been  found  as  the  cause  of  acute  or  chronic  inflammations  of 
the  pharynx  in  man.     Piana  has  observed  a  case  of  pleuritis  in  a  dog 
caused  by  an  organism  similar  to  or  identical  with  the  Leptothrix 
buccalis. 

2.  Cladothrices. — Cladothrix    asteroides    is    the    name    given    by 
Eppinger  to  an  organism  found  as  the  cause  of  pseudotubercuiar 
lesions  in  guinea-pigs.    It  is  described  as  consisting  of  filaments  with 
pseudobranches.     The  filaments  later  break  up  into  segments  of 
cuboidal  and  bacillary  shape.    The  organism  grows  rapidly  at  blood 
temperature,  poorly  under  anaerobic  conditions ;  and  does  not  liquefy 
gelatin.     It  forms  round  colonies,  which  subsequently  become  con- 

26 


402  PATHOGENIC  STREPTOTHRICES 

fluent  and  form  a  corrugated,  tenacious,  orange-colored  membrane. 
The  organism  does  not  coagulate  milk. 

Cladothrix  canis  was  found  by  Rabe  in  cases  of  peritonitis  in  dogs 
and  in  purulent  processes  of  the  skin  and  subcutaneous  connective 
tissue.  In  pus  and  softened  lymph  glands,  the  organism  appeared 
as  a  conglomeration  of  filaments.  The  latter  were  straight,  angular 
or  wavy,  branching,  and  also  found  broken  up  into  bacillary  segments. 
Rabe  considered  the  organism  the  cause  of  the  pathologic  lesions 
in  which  it  was  found.  It  is  uncertain  whether  the  organism  is  really 
a  cladothrix  or  whether  it  is  not  identical  with  the  Streptothrix  canis 
described  below. 

3.  Streptothrices. — These  have  been  found  a  number  of  times  in 
pathologic  conditions  of  domestic  animals  and  man.     In  the  latter 
Streptothrix  infection  has  been  described  by  Eppinger,  Petruschky, 
Aoyama  and  Miyamoto,  MacCallum,  Flexner,  Norris  and  Larkin, 
Wharthin  and  Olney,  Butterfield,  and  others.     The  most  interesting 
Streptothrix  infection  in  man  is  that  known  as  mycetoma,  or  madura 
foot,  of  which  two  varieties,  a  brown  and  a  white  occur.    The  latter 
is  due  to  the  Streptothrix  madurse. 

4.  Actinomyces. — The  Streptothrix  which  forms  the  typical  rosettes 
in  the  pathologic  lesions  which  it  causes  is  described  fully  in  Chapter 
XXXVII. 

PATHOGENIC  STREPTOTHRICES. 

Streptothrix  Farcinica. — The  disease  known  as  farcy  in  cattle, 
lymphangioitis  farciminosa  bovis,  farcin  du  boef  (French),  "Haut- 
wurm  des  Rindes"  (German),  was  first  described  by  Sorillon  (1829) 
as  being  frequently  seen  in  France.  Later  French  writers  have  also 
repeatedly  mentioned  it,  but  the  disease  is  now  rare  in  France.  Nocard, 
in  1887,  discovered  a  Streptothrix  as  the  cause  of  the  disease. 

The  pathologic  changes  are  generally  found  in  the  extremities  of 
the  animal,  on  the  interior  surfaces  of  which,  along  the  superficial 
veins,  non-painful  cords  and  nodules  of  very  firm  consistency  appear. 
Later  they  sometimes  become  softer,  even  fluctuating,  and  discharge 
upon  section,  a  whitish,  soft,  non-fetid  mass.  Sometimes  fluctuating 
nodules  open  spontaneously,  but  the  wounds,  as  a  rule,  soon  heal 
again.  Generally  the  lesions  do  not  become  softened,  but  remain 
permanently  composed  of  a  firm,  tough,  fibrous,  connective  tissue. 
The  disease  may  exist  a  year  or  longer  without  disturbance  of  the 
general  condition.  Later  it  occasionally  leads  to  emaciation  and 
even  cachexia  and  death. 

Farcy  in  cattle  is  caused  by  the  Streptothrix  farcinica,  which 
presents  itself  in  the  lesions  as  branching  filaments.  The  organism  is 
aerobic,  and  grows  best  between  30°  and  40°  C.  In  bouillon  it  forms 
whitish  granules  which  soon  fall  to  the  bottom  of  the  tube.  On  the 
surface  round,  dirty  gray  masses  are  found,  which  in  reflected  light 


STREPTOTHRIX  CAPR&  403 

have  a  greenish  and  dusted  appearance;  they  are  not  moistened  by 
water  which  runs  off  as  from  a  fatty  substance.  The  organism  grows 
best  in  alkaline  or  neutral  bouillon;  a  very  slight  acidity,  however, 
does  not  inhibit  development;  the  reaction  of  the  medium  is  not 
changed.  On  agar  there  develop  small,  round,  opaque  colonies  with 
thickened  margins  and  an  uneven  surface,  which  later  looks  as  if 
dusted  over.  Finally  the  surface  becomes  covered  with  a  thick, 
uneven  layer. 

The  appearance  on  coagulated  blood  serum  resembles  that  on  agar. 
The  growth,  however,  is  not  so  rapid.  On  potatoes  dry,  uneven,  pale 
yellow  colonies,  with  elevated  margins,  are  formed.  The  organism 
grows  in  milk,  but  neither  produces  acid  nor  coagulates  it.  The 
organism  is  fairly  resistant;  it  is  killed  after  an  exposure  of  ten 
minutes  to  70°  C.  When  inoculated  intraperitoneally  into  guinea-pigs 
^t  produces  pseudotuberculous  lesions;  subcutaneous  inoculations 
into  cattle  and  sheep  produce  lesions  like  those  of  farcy  found  under 
natural  conditions.  According  to  Nocard's  experiments,  rabbits,  dogs, 
cats,  and  equines  are  not  susceptible  to  the  Streptothrix  farcinica. 

Streptothrix  Canis. — A  number  of  cases  of  pleuritis  and  peritonitis 
with  slight  elevation  of  temperature  and  the  formation  of  a  reddish- 
brown,  cloudy  exudate  in  which  white  granules  can  be  seen  with  the 
naked  eye  have  been  observed  in  dogs.  The  pleura  and  peritoneum 
are  sometimes  studded  with  fibrous  appendages  or  villi,  while  the 
lungs  may  contain  hard,  gray  nodules  of  the  size  of  a  pea.  In  the 
exudate  and  the  tissue  lesions  conglomerations  of  slender  filaments 
are  found.  The  organism  has  been  studied  by  Bahr.  The  Strepto- 
thrix filaments  can  be  stained  by  Grain's  method  and  sometimes 
show  club-like  swellings  at  their  free  ends.  The  Streptothrix  canis, 
as  it  has  been  called,  grows  on  artificial  culture  media  at  incubator 
temperature,  first  only  aerobically,  later  also  anaerobically.  In  the 
depth  of  the  agar  mulberry-like  colonies  composed  of  long,  dividing 
filaments,  sometimes  thicker  at  the  ends  are  formed  after  three  or  four 
days;  threads  broken  up  into  segments  are  likewise  seen.  Pure 
cultures  inoculated  intraperitoneally  into  mice  produce  purulent 
abscesses.  Rabbits  infected  subcutaneously  develop  nodules  up  to  the 
size  of  a  hazelnut.  Dogs  can  also  be  infected  subcutaneously,  and 
the  Streptothrix  is  found  in  the  nodules  and  abscesses  which  develop. 

Actinomyces  Bicolor. — Another  Streptothrix  has  been  found  in  a  case 
of  multiple  brain  abscesses  in  a  dog.  From  the  pus  Trolldenier 
obtained  in  pure  cultures  a  Streptothrix  or  actinomyces  which  formed 
on  agar  colonies  which  were  yellow  in  the  centre  and  white  at  the 
periphery.  The  organism  which  proved  pathogenic  for  mice,  guinea- 
pigs,  rabbits,  and  dogs  was  called  Actinomyces  bicolor. 

Streptothrix  Caprae. — In  a  goat  suffering  from  a  pseudotubercular 
affection  of  the  lungs  Zschokke  found  a  Streptothrix  which  was  not 
only  Gram  positive  but  also  acid  fast.  The  organism  was  more 
thoroughly  studied  by  Silberschmidt,  who  ascribed  the  following 


404  PATHOGENIC  STREPTOTHRICES 

properties  to  it:  Very  thin,  wavy  filament,  pseudodichotomous 
division,  some  filaments  broken  up  into  segments;  on  the  surface 
filaments  broken  up  into  coccus-like  bodies  (spores).  The  organism 
grows  rapidly  under  aerobic  (not  under  anaerobic)  conditions,  both  at 
room  and  at  incubator  temperature.  The  growth  is  light  brown 
reddish  and  appears  as  if  covered  with  a  white  dust.  The  colonies  on 
agar  are  elevated  with  a  depressed  centre,  uneven,  corrugated,  and 
powdered  with  a  white  dust.  Sometimes  they  are  white  and  velvety 
and  look  like  the  growth  of  a  higher  mould.  In  bouillon  disk-like, 
dry,  isolated  colonies  are  formed  on  the  surface;  a  complete  pellicle 
does  not  form.  The  sediment  is  granular.  Growth  on  potatoes  is 
rapid ;  first  reddish,  then  white.  On  milk  a  reddish  pellicle  is  formed ; 
coagulation  does  not  occur.  Subcutaneous  inoculation  of  pure  cultures 
produce  abscesses  in  guinea-pigs,  rabbits,  and  mice.  Intraperitoneal 
or  intravenous  injections  produce  pseudotubercular  lesions. 

A  streptothrix  of  the  same  type,  also  acid  fast  and  leading  to 
pseudotubercular  lesions  in  the  lungs  of  a  young  man  suffering  and 
dying  from  diabetes,  has  been  described  by  Butterfield.  Since  the 
organism  was  not  obtained  in  pure  culture  and  no  animal  experiments 
were  made,  it  is  impossible  to  say  whether  it  is  identical  with  Strepto- 
thrix caprae  or  not. 

QUESTIONS. 

1.  What  are  the  characteristics  of  hyphomycetes  or  eumycetes? 

2.  In  what  features  do  they  differ  from  schizomycetes  ? 

3.  What  is  the  difference  between  true  and  false  branching? 

4.  What  is  the   difference  between  dichotomous  and   pseudodichotomous 
branching  or  division  ? 

5.  What  is  a  thallus? 

6.  What  is  a  mycelium? 

7.  How  does  the  mycelium  grow  and  extend? 

8.  Differentiate  between  phy  corny  cetes  and  my  corny  cetes. 

9.  What  is  the  function  of  the  fructifying  organ  of  hyphomycetes? 

10.  Name  the  four  types  of  spores  formed  by  hyphomycetes. 

11.  Describe  the  characteristics  of  the  four  different  types. 

12.  What  are  copulating  cells?    What  other  name  are  they  known  by? 

13.  What  are  suspensors? 

14.  What  is  meant  by  basipetal  conidia?    What  by  basifugal  conidia? 

15.  Why  is  chlamydospore-formation  also  called  oidium-f ormation  ? 

16.  Discuss  the  resistance  of  spores  of  hyphomycetes. 

17.  What  are  trichomy cetes ?    Why  so  named? 

18.  Name  the  four  genera  of  the  family  trichomycetes. 

19.  Describe  the  characteristics  of  the  four  genera. 

20.  Name  a  species  of  leptothrix.    Where  found? 

21.  Name  some  species  of  cladothrix. 

22.  Have  streptothricse  been  found  as  the  cause  of  human  diseases?      . 

23.  Why  are  actinomyces  considered  separately  from  streptothricse  to  which 
they  belong? 

24.  Describe  the  Streptothrix  farcinica  and  the  lesions  which  it  produces  in 
cattle. 

25.  Describe  the  morphology,  cultural  properties,  and  pathologic  lesions  of 
Streptothrix  canis. 

26.  Describe  the  morphology,  cultural  properties,  and  pathologic  lesions  of 
Streptothrix  caprse. 


CHAPTER    XXXVII. 

ACTINOMYCOSIS. 

Occurrence  and  Historical. — Actino mycosis  is  a  chronic  infectious 
disease  occurring  principally  in  cattle  and  swine,  also  in  man  and 
more  rarely  in  horses  and  sheep;  occasional  cases  have  been  observed 
in  deer,  elephants,  dogs,  and  cats.  It  is  caused  by  a  microorganism 
known  as  the  actinomyces  (actis  ray;  mykos,  fungus),  or  ray 
fungus.  Its  growth  in  the  tissues  of  susceptible  animals  leads  to 
the  formation  of  frequently  large,  granulomatous,  and  fibrous  tumor 
masses.  The  disease  in  cattle  has  been  known  for  a  long  time  as 
lumpy  jaw,  wens,  cancer  of  the  tongue,  osteosarcoma,  etc.  It  is 
now  known  that  Langenbeck,  as  early  as  1845,  saw  the  characteristic 
ray  fungi  in  a  human  affection  (then  mistaken  for  osteosarcoma).  In 
fact,  he  gave  a  picture  of  the  organism,  however,  without  recognizing 
its  nature.  In  1848  Lebert  again  furnished  drawings  of  the  ray 
fungus,  and  in  1868  Rivolta  described  it  clearly  from  a  case  of  lumpy 
jaw  in  cattle.  In  1876  Bellinger  recognized  the  parasitic  nature  of 
the  actinomyces,  and  Hartz,  a  botanist,  studied  it  from  the  biologic 
standpoint.  A  little  later  the  etiology  of  the  disease  in  man  was 
established  by  Israel.  Actinomycosis  is  distributed  over  the  entire 
world,  and  the  fungus  is  undoubtedly  very  prevalent  in  nature,  where 
it  is  found  on  grasses.  It  is  probably  always  contracted  by  the 
entrance  of  the  bearded  grains  of  barley,  oats,  wheat,  etc.,  which 
carry  the  fungus  into  a  wound  of  the  buccal  or  respiratory  mucosa.  In 
fact,  such  evidently  infected  vegetable  parts  are  very  frequently  found 
in  actinomycotic  lesions,  particularly  in  those  of  the  tongue  in  cattle. 
As  early  as  1882  Johne  observed  barley  hulls  infected  with  actino- 
myces in  the  tonsils  of  hogs,  and  he  expressed  the  belief  that  grains 
of  cereals  acted  as  the  infection  carriers  of  the  disease.  It  is  not  yet 
definitely  settled  whether  actinomycosis  in  man  and  animals  is  due 
to  the  same  species  of  ray  fungus  or  whether  there  are  several  species 
or  at  least  several  varieties.  There  is  no  evidence  that  actinomycosis 
is  ever  spread  by  direct  or  indirect  contagion  from  animal  to  animal 
or  from  animal  to  man;  it  is,  so  far  as  is  known,  only  contracted 
through  infected  vegetable  parts  gaining  entrance  into  the  tissues 
of  a  susceptible  being. 

Pathologic  Changes. — The  ray  fungus  after  entering  into  and 
multiplying  in  the  body  leads  to  a  chronic  inflammatory  reaction  with 
considerable  new  formation  of  tissue.  The  granulomatous  masses 
may  assume  a  very  large  size  and  appear  like  true  neoplasms  or 


406 


ACTINOMYCOSIS 


FIG.  161 


tumors.  It  is  claimed  that  the  ray  fungus  itself  never  leads  to  suppu- 
ration and  that  pus  appears  only  after  the  inflammatory  tissue  has 
secondarily  become  invaded  by  pyogenic  microorganisms.  In  cattle 
the  disease  generally  begins  at  the  head,  and  the  lower  jaw  is  the  part 
which  is  apparently  first  affected,  but  actually  it  is  the  posterior  part 
of  the  tongue,  the  region  of  the  papillae  circumvallatse,  which  is 
primarily  affected.  This  region  has  generally  been  found  infected 
in  every  case  of  actinomycosis  of  the  head,  and  even  when  lumpy 
jaw  had  not  yet  developed,  careful  microscopic  examination  has 
frequently  shown  invasion  of  the  tongue. 

The  most  common  tongue  affection  in  cattle  is  in  the  form  of 
ulcerations  on  the  back  of  the  tongue;  the  loss  of  substance  is  either 
round,  oval,  or  band-like  in  shape.  The  margins  of  the  ulcers  are 

elevated,  irregular,  and  ragged. 
These  ulcers  are  frequently 
covered  by  hairs  and  vegetable 
fragments.  If  a  section  is  made 
through  the  ulcerated  surface, 
grayish-white  nodules  are  seen 
extending  into  the  muscular 
portion  of  the  organ.  These 
actinomycotic  foci  contain  a 
greenish-yellow,  tenacious,  sticky 
material  in  which  may  be  seen 
grayish,  yellowish,  or  brownish 
granules,  presenting  masses  of 
the  fungi.  Sometimes  the  tongue 
contains  fistulous  tracts.  In 
other  cases  the  surface  and  the 
muscular  substance  of  the  tongue 
shows  a  smaller  or  larger  num- 
ber of  nodules,  of  peanut  to  hazel- 
nut  size.  These  have  sometimes  broken  through  the  surface  and  then 
exhibit  sharply  defined  openings.  When  such  actinomycotic  nodules 
have  existed  for  a  long  time  the  connective  tissue  of  the  tongue  is 
much  increased  and  the  organ  much  enlarged  and  very  tough  and 
hard  in  consistency,  often  as  hard  as  a  board.  This  is  the  so-called 
wooden  tongue  of  actinomycosis. 

In  the  maxillary  bones  the  disease  arises  from  the  alveoli  of  the 
teeth  or  from  the  periosteum.  When  the  alveoli  are  the  starting 
point  of  the  infection  the  normal  bone  gradually  becomes  replaced 
by  a  soft,  sarcoma-like  mass,  and  while  bone  substance  becomes 
necrotic  and  is  more  or  less  removed  new  bone  is  simultaneously 
formed  by  the  periosteum  in  consequence  of  an  osteoplastic  (bone- 
forming)  inflammatory  process.  The  new-formed  bone  may  again 
be  broken  through  by  the  granulomatous  masses  and  these  may 
protrude  externally  through  the  skin,  or  internally  into  the  cavity  of 


Actinomycosis.  Section  of  granulation  tissue 
of  jaw  of  cattle.  Carmin  stain,  Gram  gentian 
violet.  X  400.  (Author's  preparation.) 


PATHOLOGIC  CHANGES  407 

the  mouth.  In  the  interior  of  the  granulation  tissue  which  has  been 
invaded  and  partly  replaced  the  osseous  bone  substance  is  still  present 
in  the  shape  of  a  honeycombed  network  with  wide  open  spaces.  As 
the  granulation  tissue  invades  the  maxillary  bone  more  and  more  it 
often  loosens  teeth  and  lifts  them  out  of  their  alveolar  position.  The 
gums  also  become  involved  and  exhibit  larger  or  smaller  granulomatous 
masses,  which  may  ulcerate  extensively.  The  condition  of  the  maxil- 
lary bones  of  cattle  which  have  been  extensively  invaded  by  actino- 
mycosis  generally  resembles  that  of  bones  which  have  become  the 
seat  of  an  osteosarcoma,  and  hence  the  disease  was  formerly  mistaken 
for  such  a  bone  tumor.  Actinomycosis  starting  from  the  periosteum 
of  the  maxillary  bones  generally  leads  to  much  denser,  more  solid, 
more  fibrous  tumor  masses.  In  the  mouth  cavity  actinomycotic  masses 
frequently  have  the  shape  of  mushrooms  and  cauliflower  excrescences. 

FIG.  162 


Actinomycosis  of  the  inferior  maxillary  bone  of  cattle. 

Primary  actinomycosis  of  the  esophagus,  the  lower  gastro-intestinal 
tract  and  the  respiratory  tract  in  cattle,  is  rare;  the  liver,  however,  is 
frequently  full  of  actinomycotic  abscesses  in  advanced  cases. 

The  actinomycotic  tumor  of  the  head  of  cattle  after  having  grown 
to  a  certain  size  frequently  leads  to  superficial  ulcerations.  It  then 
presents  an  uneven  ulcerated  surface  from  which  granulomatous 
cauliflower  masses  may  protrude.  Such  ulcerations  discharge  a 
yellowish,  creamy,  or  very  tenacious,  gluey  pus.  In  this  pus  the 
typical  grayish-yellow  or  brownish  granules  of  the  ray  fungus  are 
generally  found. 

Subcutaneous  actinomycosis  in  cattle  is  not  always  confined  to  the 
head,  but  is  also  found  in  the  back,  legs,  and  thighs.  In  swine 
actinomycosis  frequently  finds  entrance  through  the  tonsils  or  through 
the  nipples,  the  latter  being  injured  when  the  animals  are  kept  in 


408  ACT1NOMYCOSIS 

stubble  fields.  In  horses  the  disease  has  been  observed  in  the  tongue, 
lymph  glands,  etc.  A  fibrous  cord  in  a  horse,  seen  in  1908  by  the 
author  in  a  dissecting-room  subject,  was  found  to  be  due  to  actinomy- 
cosis.  Cattle  and  hogs  likewise  contract  actinomycosis  from  castration 
wounds. 

In  man,  as  in  cattle,  the  ray  fungus  disease  generally  makes  its 
entrance  through  wounds  of  the  mouth.  It  has  been  known  to  follow 
the  habit  of  chewing  grain  or  picking  the  teeth  with  straws.  Pieces 
of  barley  and  pieces  of  straw  have  been  found  in  decayed  teeth  from 
which  the  disease  evidently  started.  In  man,  actinomycosis  frequently 
begins  in  the  intestines,  where  also  its  principal  lesions  develop  and 
from  which  place  it  generally  invades  the  liver. 

Microscopically,  parts  infected  with  actinomyces  show  a  gran- 
ulation tissue  composed  of  lymphoid  and  epithelioid  cells,  and  also 
polynuclear  giant  cells.  The  latter  are  generally  less  regular  in  out- 
line than  those  found  in  tuberculosis.  External  to  the  cellular  tissue 
a  zone  of  fibrous  tissue  is  found.  Actinomycotic  tissue  also  shows  a 
more  or  less  pronounced  infiltration,  with  ordinary  polynuclear 
leukocytes ;  basophilic  plasma  cells  are  likewise  seen. 

Systematic  Classification  and  Morphology. — The  microorganism 
causing  the  disease  actinomycosis  is  variously  classified  by  different 
authors.  Some  look  upon  it  as  a  streptothrix  and  class  it  with  the 
so-called  pleomorphous  bacteria,  others  class  it  among  the  trichomy- 
cetes  (hair  fungi),  and  still  others  place  it  in  a  class  by  itself,  inter- 
mediate between  the  bacteria  (schizomycetes)  and  the  lower  moulds 
(trichomycetes).  Quite  a  number  of  species  of  actinomyces  exist 
(generally  as  saprophytes)  in  the  outside  world,  and  only  occasionally 
invade  the  animal  tissues,  there  to  lead  a  parasitic  existence. 

Microscopic  Examination  of  Fresh  Material. — In  making  a  micro- 
scopic examination  for  the  actinomyces,  or  ray  fungus,  it  is  best  to 
scrape  some  of  the  purulent  material  from  an  actinomycotic  ulcer  or 
abscess  with  a  small  scalpel  and  to  spread  this  material  on  a  slide, 
where  it  is  covered  with  a  cover-glass  without  making  any  pressure. 
If  the  preparation  is  now  held  against  a  dark  background  the  so- 
called  actinomyces  granules  can  frequently  be  recognized  with  the 
naked  eye.  The  majority  are  from  0.1  to  0.2  mm.  in  diameter  and 
cannot  be  seen  without  magnification;  but  some  are  0.5  mm.  and 
more  in  size,  and  these  appear  as  small  yellowish,  yellowish-green,  or 
yellowish-brown  granules  to  the  naked  eye.  As  these  masses  of  fungi 
grow  larger  and  older  they  generally  appear  more  highly  colored  and 
more  pronouncedly  yellowish  brown.  If  the  preparation  is  now 
examined  under  the  low  power  of  the  microscope  the  characteristic 
rosettes  are  seen,  formed  by  club-  or  pear-shaped  bodies  arranged 
in  a  radiating  manner  around  a  common  centre.  From  this  typical 
arrangement,  always  shown  in  pus  or  tissues,  the  name  actinomyces, 
or  ray  fungus,  is  derived.  The  rosette  of  clubs  is  frequently  surrounded 
by  lymphoid  cells,  polynuclear  leukocytes,  or  giant  cells.  Sometimes 


STAINING  PROPERTIES  409 

the  fungi  and  the  tissue  cells  surrounding  them  have  undergone 
calcification,  and  the  rosette  appearance  is  then  not  so  characteristic ; 
it  may,  however,  be  brought  out  by  the  addition  of  dilute  acetic  acid, 
which  will  dissolve  the  lime  salts. 

Staining  Properties. — It  is  unnecessary  and  in  fact  disadvantageous 
to  attempt  to  prepare  stained  cover-glass  preparations,  because  in 
preparing  a  thin  spread  the  characteristic  rosette  form  is  destroyed. 
To  study  the  finer  structures  of  the  actinomyces  rosettes  it  is  necessary 
to  embed  and  section  tissues  containing  them.  The  sections  may  be 
stained  with  hematoxylin  and  eosin,  or,  better  still,  by  one  of  the 
following  methods : 

SCHLEGEL'S  METHOD. — 1.  Stain  celloidin  sections  for  four  to  five 
hours  in  the  incubator  in  a  strong  alcoholic  solution  of  eosin — the 
alcohol-soluble  eosin  must  be  used,  not  the  water  soluble  eosin. 

2.  Wash  rapidly  in  90  per  cent,  alcohol. 

3.  Stain  for  five  to  ten  minutes  in  watery  hematoxylin  solution. 

4.  Wash  in  water. 

5.  Wash  rapidly  in  alcohol. 

6.  Lift  on  slide  with  lifter. 

7.  Dry  with  filter  paper. 

8.  Pour  on  some  xylol  or  carbol-xylol  to  clear. 

9.  Mount  in  Canada  balsam. 

WEIGERT'S  METHOD. — 1.  Stain  sections  well  with  borax-alum,  or 
lithium  carmin.  Lithium  carmin  generally  stains  in  twenty  to  thirty 
minutes;  the  other  carmins  must  act  for  twenty-four  hours. 

2.  Wash  in  acidulated  alcohol  (70  per  cent,  alcohol,  100  parts  -f 
HC1  ^  per  cent). 

3.  Wash  in  95  per  cent,  alcohol. 

4.  Stain  in  anilin-water  gentian  violet  for  five  to  twenty  minutes. 

5.  Wash  in  normal  salt  solution. 

6.  Gram's  decolorizing  solution  a  few  seconds  (iodin,  1  part;  iodide 
of  potash,  2  parts;  water,  300  parts). 

7.  Decolorize  in  anilin-oil-xylol. 

8.  Wash  in  several  changes  of  pure  xylol. 

9.  Mount  in  Canada  balsam. 

MALLORY'S  METHOD. — 1.  Stain  sections  for  at  least  ten  minutes 
in  a  saturated  solution  of  water-soluble  eosin.1 

2.  Wash  rapidly  in  water. 

3.  Stain  for  a  few  minutes  in  anilin-water  gentian  violet. 

4.  Wash  in  normal  salt  solution. 

5.  Iodin  solution  (iodine,  1  part;  iodide  of  potash,  2  parts;  water, 
100  parts). 

6.  Wash  in  water,  lift  on  slide  with  a  lifter,  dry  with  filter  paper. 

7.  Decolorize  on  slide  with  anilin  oil,  several  changes. 

8.  Clear  with  xylol  several  changes. 

9.  Mount  in  Canada  balsam. 

1  The  section  may  also  first  be  stained  with  alum  carmin,  but  not  very  deeply. 


410 


ACTINOMYCOSIS 


Morphology  of  the  Actinomyces  Granule. — The  last  two  methods 
given  stain  the  filaments  blue  and  the  pear-shaped  clubs  yellow  or 
pink.  In  sections  stained  by  these  methods  the  details  of  the  structure 
of  the  rosette  can  be  studied.  The  actinomyces  granule  in  tissue  or 
pus  is  found  to  be  a  more  or  less  spherical  or  oval  body  which  includes 
in  its  interior  an  only  partially  filled  cavity.  The  wall  of  the  cavity 
is  composed  of  densely  crowded,  richly  branched  filaments,  which  on 

FIG.  163 


Actinomycosis  of  a  fibrous  cord  of  ahorse.     X  400.     (Author's  preparation.) 

the  whole  have  a  radial  direction.  The  interior  of  the  cavity  also  con- 
tains branched  filaments,  but  in  comparison  with  the  dense  wall 
only  in  moderate  numbers.  Many  of  the  filaments  are  quite  slender 
and  uniformly  stained.  They  are  provided  with  a  delicate  membrane. 
These  filaments  are  not  straight,  but  wavy;  spirillum  and  spirocheta- 
shaped  filaments  are  also  present.  The  primary  branches  divide 
dichotomously.  Lines  of  partition  are  seen  in  the  filaments;  they 
divide  them  into  shorter  segments,  bacilla-like  portions,  and  even 


CULTURAL  PROPERTIES  411 

small,  round,  cocci-like  bodies.  The  latter  are  the  spores.  They  are 
not  only  found  in  the  interior  of  the  filaments,  but  also  free  between 
them.  These,  however,  unlike  the  spores  of  the  simple,  true  bacteria, 
have  the  same  staining  properties  as  the  filaments,  but  they  are  more 
resistant  than  the  filaments  (see  below).  In  addition  to  the  slender 
well-stained  filaments,  there  are  seen  coarse,  poorly  and  irregularly 
stained  threads,  which  are  provided  with  a  coarser,  more  densely 
stained  membrane.  Very  pale  threads  containing  stained  round 
bodies,  only  here  and  there,  are  also  seen.  These  are  filaments 
in  a  well-advanced  stage  of  degeneration.  At  the  periphery  of  the 
actinomyces  granule  the  free  ends  of  the  filaments  have  formed  clubs, 
which  are  more  or  less  round  or  more  commonly  elongated  and  truly 
club-  or  pear-shaped.  These  club-shaped  formations  are  due  to  a 
gelatinous  degeneration  of  the  membrane  of  the  coarser  and  less 
uniformly  stained  filaments;  the  latter  can  often  be  seen  at  the  inner 
portion  of  the  swollen  club.  The  clubs  are  sometimes  divided  into 
segments  by  one  or  more  lines  of  partition,  and  the  individual  parts 
forming  them  are  often  bulged  out  at  the  sides,  so  that  the  lines  of 
division  appear  as  constricting  rings.  All  these  morphologic  features 
are  due  to  degenerative  changes  of  the  membrane  and  of  the  inner  proto- 
plasmic thread  of  the  filaments.  The  wall  of  the  actinomyces  granule 
or  rosette  is  deficient  in  one  place  where  the  filaments  partially  filling 
the  cavity  project  out  of  it  into  the  surrounding  pus  or  tissue.  These 
projecting  masses  of  filaments  have  been  called  the  root  of  the 
actinomyces  rosette.  All  of  these  features  are  well  marked  in  com- 
paratively young  rosettes.  When  they  grow  older  most  of  the  details 
disappear  and  often  nothing  is  left  but  a  rosette  formed  by  the 
degenerated  gelatinous  clubs.  The  best  material  for  studying  the  finer 
details  of  the  structure  of  the  actinomyces  granules  is  a  soft  granulo- 
matous  tissue,  not  a  hard,  fibrous  one,  which  generally  contains  older 
actinomyces  colonies  which  no  longer  show  the  finer  details. 

Cultural  Properties. — All  who  have  attempted  to  obtain  actinomyces 
in  pure  cultures  agree  that  this  is  a  difficult  task.  Actinomycotic 
material  must  be  obtained  from  the  interior  of  a  lesion  in  an  aseptic 
manner  and  then  numerous  culture  tubes  must  be  inoculated,  because 
in  only  a  very  small  percentage  will  the  fungus  grow  in  a  first  gener- 
ation. The  material  obtained  is  first  rubbed  up  in  a  sterile  mortar 
with  the  addition  of  melted  gelatin  or  bouillon  and  then  again  inocu- 
lated into  sterile  melted  gelatin  tubes  and  their  contents  are  finally 
poured  into  Petri  dishes.  Some  of  these  must  be  kept  under  aerobic 
others  under  anaerobic  conditions,  because  some  varieties  or  species 
of  actinomyces  are  aerobic,  others  anaerobic.  When  a  first  generation 
has  once  been  obtained  it  is  easy  to  secure  the  organism  in  a  second 
and  in  subsequent  generations  on  gelatin,  glycerin  agar,  blood  serum, 
potatoes,  and  in  hen's  and  pigeon's  eggs.  On  gelatin  plates,  if  the 
fungus  grows  in  a  first  generation  under  aerobic  conditions,  a  small, 
gray  point  appears  on  the  fifth  or  sixth  day.  Microscopic  examination 


412 


ACTINOMYCOSIS 


shows  it  to  consist  of  a  fine  network  of  filaments  radiating  from  a  com- 
mon centre.  After  several  more  days  the  colony  assumes  a  yellowish 
cloudy  appearance.  If  one  of  these  young  first  generation  colonies  is 
taken  up  with  a  strong  platinum  loop  and  rubbed  over  the  surface  of  a 
glycerin  agar  slant  and  this  is  incubated,  there  appears,  after  twenty- 
four  hours,  on  the  surface  in  patches  a  thin,  gray,  moist,  gelatinous 
film,  which  after  another  day  becomes  thicker  and  more  cloudy. 
After  a  further  lapse  of  time  whitish  points  project  above  the  sur- 
rounding surface  and  the  growth,  except  at  the  very  periphery, 
becomes  opaque.  In  older  colonies  the  former  projecting  points  have 
become  larger  button-like  masses  which  are  elevated  high  over  the 
surface  of  the  culture  medium.  These  masses  now  become  yellow  or 
yellowish  red  or  even  dark  red  (brick  red),  they  are  very  firmly  united 
with  the  culture  medium  soil  by  filaments  penetrating  into  its  depth, 
and  it  is  difficult  to  remove  them.  They  must  be  dislodged  with  a 
strong  platinum  loop  or  spatula.  The  condensation  water  of  the 
culture  tubes  remains  clear.  In  gelatin  stick  culture  there  is  an 
abundant  growth  on  the  surface,  but  little  along  the  stick-canal. 
The  description  here  given  refers  particularly  to  actinomyces  obtained 
by  Bostroem  from  persons  and  domestic  animals.  The  fungi  are 
facultative  aerobes;  they  grow  much  better  in  the  presence  of  oxygen, 
but  can  also  grow  in  the  absence  of  atmospheric  air.  Wolf  and 
Israel  have,  from  two  human  cases,  obtained  actinomyces  stems 
which  grow  very  poorly  under  aerobic  but  very  well  under  anaerobic 
conditions,  particularly  if  inoculated  into  hen's  or  pigeon's  eggs. 

FIG.  164 


Actinomycosis.    Cover-glass  preparation  from  pure  culture  showing  true  branching.     X  1000. 

(Author's  preparation.) 

Morphology  of  the  Fungus  in  Pure  Culture. — If  cover-glass  prepar- 
ations are  made  from  pure  cultures  of  actinomyces  and  stained  with 
Loeffler's  methylene  blue  or  by  Gram's  method,  slender  and  more 
coarse  filaments  which  show  true  branching  are  seen.  The  filaments 


FREQUENCY  OF  NATURAL  INFECTION  413 

are  not  stained  uniformly,  but  they  show  unstained  portions  which 
are  known  as  vacuoles.  Some  of  the  filaments  are  broken  up  into 
rod-like  portions,  others  into  round  or  oval  granules.  The  latter  are 
spores.  They  do  not  have  the  staining  properties  of  the  true  bacterial 
spore,  but  are  more  resistant  than  the  vegetative  filaments.  The 
characteristic  rosettes  seen  in  natural  infection  are  never  seen  in 
artificial  cultures. 

Resistance. — Actinomyces  cultures  are  quite  resistant  to  drying  out; 
they  are  killed,  according  to  Domec,  at  60°  C.  in  five  minutes,  but 
the  spores  require  75°  C.  for  five  minutes  before  they  are  destroyed. 
According  to  others  the  spores  can  resist  drying  out  for  six  years 
and  are  unaffected  by  75°  C.  for  fifteen  minutes,  but  killed  only  by 
80°  C.  acting  for  fifteen  minutes. 

Animal  Inoculation.— On  calves  and  small  laboratory  animals  this 
is,  as  a  rule,  not  successful.  Sometimes  a  local  self-limiting  process 
occurs,  but  a  progressing  affection  identical  with  the  natural  clinical 
course  of  the  disease  has  perhaps  never  been  produced. 

Natural  Infection. — It  is  now  generally  accepted  that  the  disease 
is  almost  invariably  transmitted  to  persons  or  susceptible  animals 
through  the  hulls  of  grain,  straw,  hay,  splinters  of  wood,  etc.,  which 
are  contaminated  with  the  fungus  and  carry  it  deep  down  into  the 
tissues.  Johne's  early  observation  in  regard  to  finding  actinomyces- 
bearing  barley  hulls  in  the  tonsils  of  swine  has  already  been  referred 
to.  Since  then  numerous  observers  have  succeeded  in  finding  such 
infected  material  in  the  tissues  of  actinomycotic  lesions  in  man 
and  animals.  Bostroem  examined  32  cases  of  actinomycosis  of  the 
upper  or  lower  jaw  in  cattle,  and  he  regularly  found  hulls  deeply 
wedged  in  between  the  teeth  and  the  gums,  or  he  found  them  still 
deeper  in  the  osseous  granulations.  These  hulls  were  studded  with 
ray  fungi.  Bang  showed  that  the  ray  fungus  grows  well  on  grains, 
particularly  on  barley.  Berestnew  succeeded  in  finding  ray  fungi  on 
dry  grasses,  grains,  and  straw  by  introducing  them  into  sand  kept  in 
glass  vessels  in  the  incubator  and  moistening  the  vegetable  parts  with 
sterile  water.  Under  these  conditions  colonies  of  ray  fungi  devel- 
oped on  the  material  collected  in  the  outside  world.  The  mode  of 
transmission  through  infected  grain  has  also  been  shown  to  be  true  of 
man  in  whom  it  has  been  repeatedly  demonstrated  that  the  entrance 
of  hulls  into  the  tissues  of  the  mouth,  the  pharynx,  the  hands,  etc., 
has  been  followed  directly  by  the  development  of  actinomycotic 
lesions.  There  is  no  convincing  evidence  of  direct  transmission 
from  one  sick  animal  to  another,  and  no  case  has  ever  been  reported 
to  show  contagion^  from  cattle  or  other  animals  to  man. 

Frequency  of  Natural  Infection. — In  man  actinomycosis  is  com- 
paratively rare;  in  cattle  it  is  very  common  and  occurs  among  them 
both  sporadically  and  endemically,  and  occasionally  as  an  epidemic. 
In  certain  localities  the  fungus  is  evidently  widespread  and  the 
opportunities  for  infection  are  numerous. 


414  ACTINOMYCOSIS 

The  spreading  in  the  tissues  along  the  lymphatics  occurs  probably 
through  the  filaments  which  protrude  from  the  rosette  cavity  at  its 
root  and  penetrate  into  the  surrounding  tissues,  in  which  small  bacilli- 
like  segments  and  spores  become  detached.  They  are  taken  up  by 
the  leukocytes,  which  later  wander  away  and,  instead  of  killing  the 
fungi  by  phagocytic  action,  perish  themselves  in  consequence  of  cell 
necrosis.  The  spores  set  free  in  this  manner  form  the  focus  for  the 
formation  of  new  actinomycotic  granules  or  rosettes.  The  spreading 
of  the  infection  by  continuity  depends  to  a  large  degree  upon  the  tissue 
reaction.  Where  much  fibrous  connective  tissue  is  formed  around 
the  actinomyces  granules,  they  are  likely  to  undergo  complete  gelatin- 
ous degeneration  with  club  formation,  and  to  die  out.  Where  there 
is  more  tissue  necrosis  and  softening  and  less  fiber  formation  the 
ray  fungi  tend  to  remain  alive,  to  form  new  young  filaments,  and  to 
spread  through  these  extending  filaments  and  their  rods  and  spores. 

Actinobacillosis. — Ligniere  and  Spitz,  in  1902,  described  a  disease 
of  cattle  in  Argentina,  which  is  said  to  occur  sporadically  and  also  in 
epizootic  form.  This  disease  was  called  actinobacillosis.  Its  symptoms 
and  pathologic  changes  are  almost  identical  with  those  of  actinomy- 
cosis  in  cattle.  The  lesions  are  most  commonly  found  in  the  sub- 
cutaneous tissue  of  the  neck;  the  tongue,  however,  has  been  found 
affected  in  only  5  per  cent,  of  the  cases;  changes  in  the  lungs,  lymph 
glands,  lymphatics,  pharynx,  udder,  and  bones  were  found  even  less 
frequently.  The  subcutaneous  changes  consist  in  diffuse  infiltrations 
with  abscess  formation  which  project  as  round  swellings  above  the 
surrounding  surface.  The  tumors  are  generally  not  very  large  but 
of  rather  moderate  size,  and  only  after  having  existed  for  weeks  and 
months  do  they  discharge  a  very  tenacious  whitish  or  greenish  pus, 
in  which  grayish-white  granules  of  the  size  of  a  pinhead  can  be  seen. 
The  cavities  of  the  abscesses  contain  a  grayish  granulation  tissue. 
The  disease,  however,  more  frequently  leads  to  the  production  of 
hard,  fibrous  connective-tissue  masses,  rather  than  causing  abscess 
and  granulation  tissue  formation.  When  the  process  invades  the 
tongue  the  picture  of  the  wooden  tongw  of  ordinary  actinomycosis  is 
produced.  Microscopic  examination  of  the  granules  found  in  pus 
shows  them  to  resemble  the  rosettes  of  ordinary  actinomycosis. 
Ligniere  and  Spitz,  however,  claim  that  if  the  granules  are  rubbed 
up  in  a  mortar  and  inoculated  into  culture  media,  a  growth  of  bacilli 
not  much  larger  than  1  to  1.8  micron  long  and  0.4  wide  is  formed. 
Inoculations  of  these  organisms  into  cattle  are  said  to  have  produced 
lesions  like  those  found  under  natural  conditions. 


QUESTIONS  415 


QUESTIONS. 

1.  What  is  the  technical  name  for  lumpy  jaw  in  cattle?     What  organism 
causes  it? 

2.  For  what  pathologic  process  was  the  disease  formerly  generally  mistaken, 
and  why? 

3.  Who  were  the  investigators  who  first  saw  the  ray  fungus?    Who  first  recog- 
nized its  significance? 

4.  Where  does  the  ray  fungus  exist  in  the  outside  world? 

5.  To  what  changes  does  it  lead  after  invading  the  tissues  of  susceptible 
animals? 

6.  Describe  the  actinomycotic  tongue  affections  in  cattle.     Why  called  under 
some  circumstances  the  wooden  tongue? 

7.  Describe  the  spread  of  the  disease  in  the  maxillary  bones;  also  the  final 
appearance  of  an  advanced  case. 

8.  What  is  an  osteoplastic  inflammation? 

9.  What  is  meant  by  necrosis  of  the  bone? 

10.  Which  internal  organ  in  cattle  is  frequently  the   seat  of  actinomycotic 
lesions  in  advanced  cases? 

11.  What  animals  besides  cattle  are  susceptible  to  actinomycosis? 

12.  What  lesions  does  actinomycosis  produce  in  man? 

13.  Discuss  the  systematic  classification  of  the  ray  fungus. 

14.  Describe  the   process  for  the  microscopic  examination  of  actinomycotic 
granulation  tissue  or  pus. 

15.  Describe  the  ray  fungus  as  generally  seen  in  pus. 

16.  Describe  some  methods  of  staining  the  ray  fungus  in  celloidin  sections. 

17.  Describe  the  finer  details  of  an  actinomyces  granule  as  it  can  be  studied  in 
a  section. 

18.  What  are  the  clubs  of  the  ray  fungus  which  form  the  rosette? 

19.  How  is  a  first  culture  from  actinomycotic  material  obtained? 

20.  Describe  the  cultural  properties  of  the  ray  fungus. 

21.  Describe  the  appearance  of  the  ray  fungus  in  a  stained  cover-glass  prepar- 
ation obtained  from  a  pure  culture. 

22.  Discuss  the  resistance  of  the  ray  fungus. 

23.  What  animals  are  very  susceptible  to  artificial  ray  fungus  inoculation? 

24.  How  is  actinomycosis  generally  contracted  by  cattle,  horses,  hogs? 

25.  How  is  it  generally  transferred  from  cattle  to  man? 

26.  How  can  the  presence  of  actinomycosis  on  grasses  be  sometimes  demon- 
strated? 

27.  How  does  the  ray  fungus  spread  after  it  has  once  penetrated  into  the 
tissues? 


CHAPTEE    XXXVIII. 

HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE— LEECHES, 

OR    BURSATTEE  —  PNEUMONOM YCOSIS  —  DERMATOMYCOSIS— 

TRICHOPHYTON  TONSURANS— ACHORION    SCHONLEINII 

— FUSARIUM  EQUINORUM— OIDIUM  ALBICANS. 

IN  the  preceding  two  chapters  the  diseases  due  to  trichomycetes, 
including  streptothrix  and  actinomyces  infections,  have  been  con- 
sidered. These  organisms  belong  to  the  lower  hyphomycetes,  but 
the  higher  hyphomycetes  are  likewise  the  cause  of  some  animal 
diseases. 

LEECHES,  OR  BURSATTEE. 

Under  the  name  of  leeches,  bursattee,  summer  sore,  hyphomykosis 
destruens  equi,"Bosartige  Schimmel-Krankheit  der  Pferde"  (German), 
a  disease  of  horses  apparently  due  to  hyphomycetes  has  been  described. 
Whether  the  disease  found  in  India  and  other  parts  of  Asia  is  absolutely 
identical  with  the  similar  affection  reported  in  the  United  States  is  a 
question  not  fully  settled.     The  name  bursattee  is  derived  from  the 
Indian  word  burus,  rain,  because  it  was  believed  that  some  causal 
relation  existed  between  the  disease  and  the  rainy  season  in  India. 
The  disease  in  India  has  been  described  by  F.  Smith  and  Steel  and 
in  the  Sunda  Islands  by  DeHaan  and  Hoogkamer.    The  pathologic 
lesions  there  noticed  consist  in  very  firm  nodules  below  the  skin, 
particularly  in  the  lips,  the  alse  of  the  nose,  the  eyelids,  but  also  on 
the  body  and  the  extremities  and  in  the  mucosa  of  the  mouth  and 
•nasal  cavities.     The  nodules  in  the  mucosa  later  change  into  open 
ulcers  covered  by  an  easily  bleeding  granulation  tissue  surrounded 
by  ragged,  uneven  margins.     The  ulcers  burrow  deep  down  in  the 
tissue,  invade  the  bone,  sometimes  perforate  it,  and  form  fistulous 
tracts.     In  the  latter  and  in  the  granulation  tissue  grayish-yellow 
masses  or  plugs  are  found,  firmly  adherent  to  the  surrounding  tissue 
and  sometimes  calcified.     In  these  masses  the  mycelia  and  spores  of 
a  mould  are  found,  which  was  obtained  by  DeHaan  in  pure  culture. 
Inoculation  experiments,  however,  failed  to  reproduce  the  disease. 
As  it  occurs  in  the  United  States  the  disease  was  first  more  fully 
described  by  Neal,  of  Florida,  who  noticed  it  in  equines  and  also  in 
cattle  which  had  spent  much  time  in  ponds  and  swamps.    Dawson,  of 
Florida,  has  stated  that  leeches,  or  bursattee,  in  Florida  is  a  common 
disease  characterized  by  the  formation  of  tumor-like  masses  with 


PNEUMONOMYCOSIS  417 

some  of  the  features  of  actinomycosis.  In  the  granulation  tissue 
yellow  bodies  with  root-like  projections  are  found,  called  leeches 
by  the  natives.  These  bodies  consist  of  the  mycelium  of  the  fungus. 
Dawson  saw  the  disease  in  horses  only,  not  in  cattle.  Fish  examined 
tissues  containing  the  fungus  and  described  the  latter;  he  did  not, 
however,  obtain  it  in  pure  cultures.  The  mould  is  seen  in  the  tissues 
in  the  form  of  filaments  which  sometimes  have  club-shaped  ends  and 
some  of  which  show  septa.  The  mycelium  is  branched  and  small, 
round  bodies;  evidently  spores  are  found  in  it.  The  mycelium  is 
often  embedded  in  a  calcified  matrix  which  is  formed  in  consequence 
of  the  inflammatory  reaction  of  the  tissue. 


PNEUMONOMYCOSIS. 

Infection  of  the  lungs  of  man  and  animals  by  hyphomycetes  or 
moulds  is  comparatively  rare,  but  a  sufficient  number  of  cases  have 
been  reported  to  show  that  such  infections  do  undoubtedly  occur. 
In  animals  they  are  generally  observed  among  those  kept  in  warm, 
moist,  poorly  ventilated  places  and  fed  on  mouldy  feed.  Such  mould 
infections  of  the  lungs  are  most  frequently  seen  in  domestic  birds; 
pigeons  appear  to  be  most  susceptible,  but  chickens,  ducks,  and  geese, 
as  well  as  parrots  and  birds  in  zoological  gardens,  may  also  be  infected. 
The  disease  is  less  frequently  found  in  mammals.  Schiitz,  Rivolta, 
Martin,  Bellinger,  Lucet,  and  Peck  have  reported  cases  in  horses. 
Roeckl,  Piana,  Bournay,  Ravenel,  and  Hartenstein  in  cattle;  others 
in  sheep,  deer,  and  dogs. 

The  pathologic  changes  produced  by  the  inhaled  and  multiplying 
moulds  consist  in  the  production  of  dirty  yellow,  greenish,  mouldy 
looking  patches,  which  lead  to  ulceration,  with  plugging  up  of  the 
lumen  of  bronchioles  and  the  formation  of  catarrhal  foci  in  the 
pulmonary  parenchyma.  These  may  be  purulent,  caseous,  or  mortar- 
like  in  character;  sometimes  they  are  surrounded  by  a  fibrous  capsule 
or  infiltrate  the  neighboring  tissue  in  a  diffuse  manner.  The  moulds 
causing  these  changes  are  found  in  the  patches  and  in  the  interior 
of  the  pathologic  foci.  Microscopic  examination  is  always  necessary, 
as  some  of  the  mycotic  lesions  closely  resemble  tubercular  changes 
and  in  horses  they  have  resembled  glanders. 

The  moulds  which  have  been  found  in  cases  of  pneumonomycosis 
are  the  following:  Aspergillus  fumigatus  and  Aspergillus  nigrescens 
form  a  colorless  mycelium  from  which  straight,  unbranched  fruit  bearers 
arise.  On  their  upper  ends  the  fruit  bearers  carry  a  columella  and 
a  sporangium  which  produces  numerous  spores  (conidia)  arranged  in 
radial  rows.  On  bread,  Aspergillus  fumigatus  forms  a  bluish-green 
and  later  ashy-gray  mould;  Aspergillus  nigrescens  forms  a  blackish- 
or  chocolate-brown  growth.  Aspergillus  fumigatus  is  the  mould  most 
commonly  found  in  mycosis  in  birds.  Birds  can  easily  be  infected 
27 


418     HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE 

experimentally  by  keeping  them  for  a  short  time  in  a  closed  space 
where  the  air  contains  spores  of  this  mould.  Other  moulds  which 
have  occasionally  been  found  as  the  cause  of  pneumonomycosis  and 
other  internal  affections  are  Aspergillm  flavus,  niger,  and  subfucus; 
Mucor  rhizopodiformis .  and  corymbifer.  All  of  these  when  injected 
intravenously  into  rabbits  in  an  emulsion  containing  numerous  spores, 
produce  multiple  mycotic  foci  in  the  internal  organs  from  which  the 
animals  die.  Natural  infection  with  them  are,  however,  rare. 


DERMATOMYCOSES. 

A  variety  of  diseases  of  the  skin  due  to  hyphomycetes  or  moulds 
occur  in  man  and  the  domestic  animals.  The  most  important  of  the 
microorganisms  which  cause  such  affections,  known  under  the 
collective  name  of  dermato mycoses,  are  the  following: 

Trichophyton  Tonsurans. — This  mould  is  the  cause  of  the  skin 
disease  known  as  herpes  tonsurans,  characterized  by  the  formation 
of  scales  and  the  falling  out  of  the  hair.  It  has  been  found  associated 
with  this  affection  in  man,  the  horse,  dog,  cat,  goat,  sheep,  and  hog. 
It  was  discovered  by  Gruby  (1842)  and  Malmsten  (1846). 

METHOD  OF  EXAMINATION. — In  order  to  examine  this  and  other 
moulds  causing  various  dermatomycoses,  some  scales  with  attached 
hairs  must  be  removed  from  the  skin  by  scraping  with  a  scalpel. 
The  material  is  then  best  placed  in  a  test-tube  and  well  shaken  for 
some  time  with  chloroform,  in  order  to  extract  the  fat.  After  this  has 
been  accomplished,  the  chloroform  is  poured  off  and  its  last  remnants 
are  allowed  to  evaporate  from  the  scales  and  hairs.  The  latter  are 
then  placed  on  a  slide  soaked  in  a  33^  per  cent,  solution  of  caustic 
soda  or  potash,  covered  with  a  cover-glass  and  examined  in  this  fluid 
under  a  higher  power  dry  lens  of  the  microscope.  The  scales  and 
hairs  are  immersed  in  the  strong  alkaline  solution  in  order  to  make 
them  transparent  so  that  the  filaments  and  the  spores  of  the  mould 
may  become  readily  visible.  They  are  then  seen  surrounding  the 
hair  as  a  septate  mycelium  and  as  rows  of  highly  refractive  round 
bodies  which  are  the  spores.  So  many  of  the  latter  are  usually  present 
that  it  is  often  impossible  to  detect  the  filaments;  they  can,  however, 
generally  be  more  easily  seen  in  the  scales  than  in  the  hairs.  The 
spores,  as  a  rule,  measure  4  to  6  micra  in  diameter,  but  they  may  vary 
between  2  to  8  micra.  The  hyphen  or  filaments  are  about  4  micra 
thick.  Pure  cultures  are  difficult  to  obtain,  since  the  hair  and  scales 
of  skin  also  contain  numerous  bacteria.  Sabouraud  has  succeeded 
in  getting  a  growth  of  the  Trichophyton  tonsurans  in  a  beerwort 
medium  composed  of  maltose  4  parts,  peptone  2  parts,  tinctura  fucus 
crispa  1.5  parts,  and  water  to  make  100  parts.  Krai's  method  consists 
in  rubbing  up  the  hairs  with  sterile  silicon  powder  and  inoculating  it 
in  gelatin  tubes,  from  which  plates  are  poured.  Kitt  succeeded  in 


TRICHOPHYTON  TONSURANS 


419 


washing  the  scales  and  hairs  in  an  alkaline  (potassium  hydroxide) 
solution,  which  killed  the  bacteria,  but  left  enough  mould  intact  for 
subsequent  successful  inoculations.  The  trichophyton  grows  best  at 


Fio.   165 


Portion  of  a  hair  invaded  by  the  Trichophyton  endo-ectothrix.  X  550.  a,  a,  chains  of 
spores  in  focus;  &.  a  chain  situated  farther  within  the  hair,  and  hence  not  in  focus.  (P'rom  a 
photomicrograph.) 

i    FIG.  166 


Trichophyton  ectothrix  culture,  three  weeks  old,  from  a  case  of  Tinea  sycosis.     (Mewborn.) 


420     HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE 

about  30°  C.  It  liquefies  gelatin,  on  which  it  forms  a  white,  dusty  cover 
which  is  difficult  to  break  up.  On  potatoes  a  folded  felty,  sometimes 
white,  at  other  times  yellowish,  reddish,  or  dark  membrane  is  formed. 
If  examined  microscopically  the  growth  shows  a  septate,  colorless 
mycelium,  and  round  or  oval  chlamydospores.  Different  cultures 
vary  considerably  in  certain  features,  so  that  a  number  of  varieties 
have  been  distinguished,  such  as  Trichophyton  megalosporon  (large 
spores),  Trichophyton  microsporon  (small  spores),  and  several 
others.  Spores  in  pure  cultures  are  generally  formed  within  the  hyphen 
as  chlamydospores.  In  the  skin  and  hairs  they  are  formed  by  the 
breaking  up  of  the  filaments  into  segments  (gemmse). 

FIG.  167 


Portion  of  a  hair  showing  the  Microsporon  Audouini.     (From  a  photomicrograph.) 

One  of  the  varieties  of  the  Trychophyton  tonsurans  has  been 
called  Microsporon  Audouini,  and  two  of  its  subvarieties,  the  equinum 
and  the  caninum,  have  been  found,  respectively,  in  horses  and  dogs. 
Certain  authors,  however,  claim  that  the  different  varieties  of  Tricho- 
phyton tonsurans  are  really  a  single  species  producing  practically 
the  same  infection,  only  somewhat  modified  as  to  the  species  of 
animal  infected.  The  mode  of  infection  consists  in  that  the  mould 
first  penetrate  into  the  hair  roots,  where  they  multiply  and  surround 
the  hair  with  a  complete  mantle  and  then  enter  into  its  interior. 
Trichophyton  tonsurans  infections  have  also  been  observed  a  few 
times  in  domestic  birds. 

Achorion  Schonleinii. — This  mould  is  the  cause  of  the  disease 
known  as  favus,  dermatomycosis  achorina,  tinea  favosa,"Wabengrind" 
or  "Erbgrind  der  Saugethiere"  (German).  It  occurs  in  man,  mice, 
rats,  cats,  dogs,  and  rabbits.  A  few  cases  which  were  described  as 
appearing  in  horses  and  cattle  were  probably  trichophyton  infections. 
Favus  infection  is  characterized  by  the  formation  of  scaly  crusts, 
which  are  depressed  in  the  centre  and  sulphur  yellow,  at  least  in  the 
interior,  where  the  color  has  not  been  changed  by  external  influences. 


ACHORION  SCHONLEINII 


421 


The  mould  is  found  in  the  scales  of  the  skin,  in  the  form  of  homo- 
geneous or  granular  septate  and  branched  hyphens,  3  to  5  micra 
thick.  The  filaments  are  often  thinned  out  at  their  free  ends  and 
thickened  at  the  points  where  branches  arise.  Some  of  the  hyphens 


FIG.  168 


FIG.  169 


Culture  three  weeks  old  from  ringworm  of  cat 
contracted  from  ringworm  of  girl's  face.  (Mew- 
born.) 


Culture  of  Achorion  Schonleinii. 
(Mewborn.) 


are  broken  up  into  oval  spores  3  to  6  micra  in  diameter.  Sometimes 
the  mould  is  found  in  the  skin  as  a  very  dense,  felted  mycelium. 
Achorion  Schonleinii  requires  more  proteid  material  for  its  growth  on 
artificial  media  than  Trichophyton  tonsurans.  Its  optimum  temper- 


FIG.  170 


Achorion  Schonleinii:  a,  spores;  b,  c,  sporophores.     (After  Cornil  and  Ranvier.) 

ature  is  at  25°  C.  On  gelatin  its  growth  somewhat  resembles  tricho- 
phyton,  but  the  medium  is  only  liquefied  after  several  weeks.  On 
potatoes  and  beets  an  elevated,  folded,  grayish-white  growth  is  formed, 
which  later  becomes  grayish  yellow.  It  grows  well  on  agar  kept  in 


422     HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE 

the  incubator.  Artificial  cultures  of  this  mould  exhibit  considerable 
pleomorphism,  and  this  has  led  to  the  establishment  of  several  varieties. 
Achorion  infects  younger  persons  or  animals  more  easily  than  older 
ones.  Mice  are  very  frequently  infected,  and  these,  when  caught  by 
cats,  infect  the  latter.  Man  is  infected  frequently  from  animals. 

Achorion  Keratophagus. — This  organism  was  named  and  described 
by  Ercolani.  It  is  of  the  achorion  type  and  was  alleged  to  have 
been  the  cause  of  an  onychomycosis  of  donkeys  and  mules.  The 
affection  is  characterized  by  the  formation  of  cavities  between  the 
hoof  and  the  lamina  sensitiva  and  the  invasion  of  the  former  by  the 
mould.  It  has  not  been  possible  to  produce  the  disease  artificially 
by  inoculation. 

Lopophyton  Gallinarum. — This  organism,  also  known  as  Derma- 
tomyces  or  Epidermophyton  gallinarum,  is  a  variety  of  Achorion 
Schonleinii  and  causes  favus  or  tinea  cristae  galli  in  chickens.  The 
organism  affects  the  comb  and  forms  on  it  whitish,  mouldy  looking 
spots  which  finally  cover  the  entire  comb  with  a  white  layer  which 
increases  in  thickness  until  it  finally  forms  a  crust  up  to  8  mm.  thick. 
Two  types  of  hyphens  are  found  in  the  lesions,  those  of  the  one  are 
long  and  wavy,  2  to  5  micra  thick,  with  irregular  side  branches;  the 
others  are  short,  straight,  or  curved  filaments,  sometimes  dichotomously 
divided,  composed  of  three  to  four  thick- walled  segments,  which  con- 
tain a  highly  refractive  protoplasm.  The  hyphens  of  this  type  are 
from  4  to  6  micra  thick  and  later  fall  apart.  Some  investigators 
have  looked  upon  the  segments  as  spores.  Cultures  of  this  variety 
of  achorion  found  on  chickens  grow  slowly  and  form  an  intensely 
red  pigment;  they  liquefy  gelatin. 

Fusarium  Equinum. — This  name  was  given  by  Noergaard  to  a 
fusarium1  found  by  Melvin  and  Mohler  as  the  apparent  cause  of  a 
dermatomycosis  in  many  hundred  horses  in  the  Umatilla  Indian 
Reservation  in  Oregon.  Fusaria  are  hyphomycetes  with  septate 
mycelia  (mycomycetes)  and  belong  to  the  class  of  ascomycetes.  These 
are  moulds  which  develop  an  ascus  or  sac  in  which  endogenous  spores 
are  formed.  The  spores  formed  in  such  a  special  spore  sac  or  ascus 
are  known  as  ascospores.  This  organism,  it  appears,  enters  the  hair 
follicles,  where  it  multiplies,  bringing  about  an  irritation  which  causes 
pruritus  and  the  formation  of  a  little  scurf  around  two  or  three  hairs. 
When  the  scurf  is  rubbed  off  a  red,  moist,  denuded  surface  is  left 
in  its  place.  The  spores  of  the  organism  can  be  seen  in  sections  of 
the  skin.  They  are  especially  numerous  in  the  hair  follicles;  mycelial 
threads  are  likewise  encountered.  Melvin  and  Mohler  succeeded  in 
obtaining  the  organism  in  pure  cultures  and  found  that  the  growth 
remained  pure  white  on  all  media  except  plain  Dunham's  solution 
on  which  the-lower  surface  in  contact  with  the  fluid  became  a  chocolate 


1  It  is  described  fully   in  the  Twenty-fourth  Annual   Report  of    the    Bureau    of    Animal 
Industry,  1907,  Dermal  Mycosis,  etc.,  Melvin  and  Mohler,  p.  260. 


FUSARIUM  EQUINUM 


423 


color.  In  cigar  the  growth  first  assumes  a  salmon-pink  color,  but  later 
becomes  white.  Potato  is  a  very  favorable  medium  for  the  organism. 
The  frost-like  film  which  is  formed  in  three  to  four  days  on  culture 


FIG.  171 


Conidia  and  mycelium  of  Fusarium  equinum  cultivated  from  root  of  hair  of  horve  affected 
with  dermal  mycosis.     (Melvin  and  Mohler." 

FIG.  172 


Photomicrograph  of  a  five-day-old  colony  of  the  fusarium  on  an  ag  ir  plate. 
(Melvin  and  Mohler.) 


424     HIGHER  HYPHOMYCETES  AS  THE  CAUSE  OF  DISEASE 

media  is  composed  of  septate  branching  hyphens,  or  filaments.  The 
organism  forms  microconidia,  macroconidia,  and  chlamydo  spores. 
The  large  falcate,  crescentic,  or  sickle-shaped  macroconidia  or  spores, 
from  25  to  55  micra  long  and  2J  to  4J  micra  wide,  are  typical  for 
all  fusaria;  they  commence  to  germinate  in  form  three  to  ten  hours 
after  being  transplanted  into  a  favorable  new  culture  medium.  From 
a  peculiar  skin  lesion  in  a  hog,  Hart  isolated  a  culture  of  fusarium 
which  seems  to  be  identical  with  the  above  Fusarium  equinum. 
Peters  found  Fusarium  moniliforme,  discovered  by  Sheldon,  on  corn 
as  the  cause  of  a  disease  of  horses  in  which  they  lose  their  hair  and 
hoofs.  The  same  organism  is  also  said  to  have  caused  loss  of  hair  in 
cattle  and  hogs  and  of  feathers  in  chickens. 

Oidium  Albicans. — The  disease  known  as  thrush,  soor,  or  stomatitis 
oidica  is  an  inflammation  of  the  mucous  membrane  of  the  mouth  and 
pharynx.  It  occurs  in  children,  domestic  birds,  and  also  calves  and 
other  young  domestic  mammals. 

The  pathologic  lesions  consist  in  the  formation  of  grayish-white 
points  and  smaller  or  larger  dots  or  even  quite  extensive  pseudo- 
membranes.  The  lesions  later  become  brownish.  If  the  pseudo- 
membranes  are  removed  a  slightly  reddened  but  otherwise  unchanged 
mucous  membrane  is  seen.  The  pseudomembranes  are,  however, 
quite  firmly  adherent.  If  examined  microscopically  they  are  found 
to  consist  of  desquamated  swollen  epithelia  mixed  with  numerous 
wavy  or  straight  filaments  and  oval  bodies.  These  formations  are 
the  mycelium  and  spores  of  the  fungus  which  causes  the  disease  and 
which  is  known  as  Oidium  albicans  or  Monilia  Candida.  The  fungus 
is  widespread  in  air,  water,  and  on  decaying  vegetable  matter  as  a 
saprophyte,  and  only  occasionally  infects  the  buccal  and  pharyngeal 
membranes  of  young  beings.  The  mycelia  are  composed  of  cylindrical 
cells,  1  to  4  micra  wide  and  10  to  20  micra  long.  The  filaments  show 
branching  and  the  outer  ends  are  rounded  off  or  club-shaped.  The 
clubs  often  contain  oval,  highly  refractive  bodies,  the  gonidia,  or 
spores,  which  are  also  found  free  between  the  filaments.  The  free 
spores  in  air  and  food  come  in  contact  with  the  buccal  or  pharyngeal 
mucosa,  and  if  there  are  slight  epithelial  defects  the  gonidia  may 
develop  and  lead  to  the  formation  of  thrush  spots  and  membranes. 
Cases  have  also  been  observed  in  children  in  which  the  fungus  had 
penetrated  deeper  and  led  to  metastases  in  the  internal  organs.  The 
pseudomembranes  are  best  examined  in  very  dilute  acetic  acid  (1  to  3 
per  cent.),  which  brings  out  the  mycelia  and  spores  under  the  micro- 
scope. 

The  organism  can  be  easily  cultivated  in  artificial  cultures.  It  is 
best  to  use  distinctly  acid  media,  because  bacteria  will  not  easily 
develop  on  them,  but  Oidium  albicans  does.  Fresh  disks  of  apples, 
obtained  after  washing  the  apples  externally  and  dividing  them  with 
a  sterile  knife,  form  a  good  culture  medium  for  this  fungus.  It  also 
grows  on  agar,  gelatin,  coagulated  blood  serum,  and  potatoes.  The 


QUESTIONS  425 

colonies  on  gelatin  plates  are  milk  white,  on  potatoes  yellowish  or 
grayish  white,  and  give  off  an  odor  of  sour  beer.  The  growth  in 
gelatin  stick  cultures  shows  the  nail  form;  the  medium  is  not  liquefied. 
The  development  on  all  these  media  occurs  at  room  temperature, 
more  rapidly  in  the  incubator.  The  organism  on  media  containing 
sugar  assumes  a  yeast-cell  type,  while  on  media  which  contains  no 
sugar  a  mycelium  is  formed.  If  the  organism  is  inoculated  into  the 
crops  of  pigeons  the  typical  thrush  lesions  are  produced.  If  injected 
intravenously  into  rabbits  multiple  metastatic  foci  of  infection  are 
produced  and  the  animals  die.  Young  pigeons  can  be  inoculated 
in  the  mouth,  where  thrush  lesions  are  formed.  Older  pigeons  and 
chickens  are  generally  resistant. 

In  artificial  cultures  Oidium  albicans  and  other  oidia  often  appear 
as  individual  cells  and  multiply  by  budding;  hence  they  are  evidently 
closely  related  to  the  budding  fungi,  or  blastomycetes,  discussed  in 
the  next  chapter. 

QUESTIONS. 

1.  What  is  leeches  or  bursattee?    In  what  countries  encountered? 

2.  What  are  the  pathologic  lesions  of  the  disease? 

3.  Describe  the  mould  found  in  the  pathologic  changes. 

4.  What  is  meant  by  pneumonomycosis  ?    In  what  animals  does  it  occur  and 
under  what  conditions? 

5.  What  moulds  cause  pneumonomycosis? 

6.  What  is  meant  by  dermato mycosis  ? 

7.  What  mould  causes  herpes  tonsurans? 

8.  Describe  the  lesions  in  herpes  tonsurans  and  the  method  of  finding  the 
mould  causing  it. 

9.  How  does  it  look  under  the  microscope? 

10.  Describe  pure  cultures  of  Trichophyton  tonsurans  and  the  methods  of 
obtaining  them. 

11.  In  what  animal  does  the  Microsporon  Audouini  cause  skin  disease? 

12.  What  are  the  lesions  of  favus?    What  organism  causes  this  skin  affection? 

13.  Describe  the  favus  mould. 

14.  Describe  its  cultural  properties. 

15.  What  lesions  are  caused  by  Achorion  keratophagus ? 

16.  What  is  Lopophyton  gallinarum?    Describe  the  lesions  it  produces. 

17.  What  is  the  Fusarium  equinum?    Describe  the  lesions  it  produces. 

18.  What  variety  of  spores  does  the  organism  form?    What  type  is  character- 
istic for  the  genus  fusarium? 

19.  Describe  the  morphologic  and  cultural  properties  of  the  organism^ 

20.  Where  has  Fusarium  monilif orme  been  found  ? 

21.  What  organism  causes  thrush? 

22.  Where  and  in  what  animals  is  this  affection  found? 

23.  What  lesions  does  it  produce? 

24.  What  is  the  other  name  for  Oidium  albicans? 

25.  Describe  its  morphology. 

26.  How  will  you  look  for  it  in  the  lesions  of  thrush? 

27.  Describe  the  cultural  properties  of  Oidium  albicans. 

28.  To  what  organisms  is  oidium  related  ? 


CHAPTER    XXXIX. 

BLASTOMYCES— EPIZOOTIC  LYMPHANGITIS  IN  HORSES— 
BLASTOMYCOTIC  DERMATITIS. 

BLASTOMYCES. 

THERE  is  a  large  class  of  low  vegetable  microorganisms  of  which 
the  common  brewer's  and  baker's  yeast  are  the  best-known  types. 
A  few  of  these  have  been  found  as  the  cause  of  disease  in  man  and 
the  lower  animals.  The  classification  of  these  fungi  has  been  very 
difficult,  and  botanically  they  have  been  defined  as  ascomycetes, 
because,  like  these,  they  generally  form  a  number  of  endogenous 
spores  in  a  spore  sac  or  ascus.  Yeast  cells,  however,  frequently 
present  themselves  as  simple,  more  or  less  globular,  strictly  unicellular 
organisms,  and  in  them  the  whole  cell  becomes  the  ascus,  or  spore 
sac,  in  which  the  endogenous  ascospores  are  formed.  As  Lafar  has 
pointed  out,  the  term  saccharomyces  (often  improperly  used  in  path- 
ology) should  be  reserved  for  the  sugar-fermenting  organisms  of  this 
type,  since  the  word  means  sugar  fungi.  On  the  other  hand,  certain 
organisms  of  this  class  have  never  been  known  to  form  spores,  and 
they  cannot  consistently  be  classified  as  ascomycetes.  It  appears, 
however,  that  they  have  the  common  property  of  forming  buds,  i.  e., 
one  or  more  nipple-  or  sac-like  or  globular  offshoots  or  out-growths, 
which  later  become  separated  from  the  main  portion  by  a  partition 
wall  or  septum,  drop  off  and  form  new  independent  unicellular 
organisms.  This  process  of  multiplication  is  known  as  budding,  hence 
the  organisms  which  show  this  mode  of  multiplication  are  known  as 
blastomycetes,  or  budding  fungi.  Most  of  the  latter  can  also  multiply 
by  sporulation,  because  they  form  more  than  one  spore — up  to  eight 
and  higher.  Those  blastomycetes  which  do  not  form  spores  are 
grouped  under  the  genus  Torula. 

When  blastomycetes  are  budding  the  newly  formed  buds  are  not 
always  necessarily  cut  off,  so  that  the  unicellular  type  is  preserved. 
Under  some  conditions  both  the  mother  cell  and  the  bud  elongate, 
become  elliptical  or  cylindrical,  and  adhere  together.  The  process  is 
repeated  a  number  of  times  at  the  outer  ends  of  the  connected  cells, 
and  there  are  formed  in  this  manner  filaments  and,  indeed,  a  structure 
with  branching  effects  much  like  the  mycelia  of  the  hyphomycetes. 
It  has  been  ascertained  that  the  saccharomycetes  and  probably  the 
blastomycetes  in  general  possess  a  nucleus  with  a  nuclear  membrane 
and  nucleolus.  These  structures  can  only  be  seen  exceptionally  in 


BLASTOMYCES 


427 


unstained  specimens,  and  even  in  stained  specimens,  they  are  exhibited 
with  difficulty;  in  young,  resting  cells,  and  by  special  staining  methods. 
To  exhibit  the  nuclear  structures,  the  yeast  cells  must  first  be  fixed 
in  picric  acid,  and  after  having  washed  this  out  they  must  be  stained 
with  Haidenhain's  iron-hematoxylin.  It  has  also  been  shown  that 
the  nucleus  of  the  yeast  cell  divides  by  a  simple  karyokinetic  or  by  an 
(un/totic  type  when  a  bud  is  produced  or  when  the  endogenous  asco- 


FIG.  173 


Pseudofarcy,  or  blastomycosis,  in  a  Filippino  pony.     The  picture  shows  several  swollen, 
subcutaneous  lymph  nodes  and  one  open  ulcer.     (Strong.) 


spores  are  formed.  The  latter  are  generally  somewhat  more  resistant, 
particularly  to  drying  out,  than  the  adult  vegetative  organism.  In 
addition  to  the  nucleus  the  vegetative  adult  organism  also  often  exhibits 
much  more  readily  seen  vacuoles  filled  with  fluid  and  in  old  cells 
highly  refractive  granules,  which  consist  of  proteid  material,  but  also 
contain  some  fat.  In  cover-glass  preparations  the  blastomyces  can 
be  stained  by  the  ordinary  watery  anilin  stains;  in  tissues  they  are  best 


428  PSEUDOFARCY,  OR  EPIZOOTIC  LYMPHANGITIS 

exhibited  by  the  eosin-alkaline  methylene-blue  method  of  Mallory. 
In  artificial  cultures  they  often  form  thick  membranes  or  pellicles  on  the 
surface  of  the  culture  medium  and  some  of  them  form  zoogleal  masses. 


PSEUDOFARCY,  OR  EPIZOOTIC  LYMPHANGITIS. 

Occurrence  and  Pathologic  Lesions. — Under  the  above  names  a  disease 
of  horses,  which  is  also  known  as  saccharomycosis,  or  blastomycosis 
farcimihosus,  has  been  described.  Clinically  it  resembles  the  cutaneous 
type  of  glanders,  or  farcy,  and  has  therefore  been  called  pseudofarcy. 
It  was  first  described  by  Italian  and  French  observers;  later  it  was 
reported  from  Japan  and  other  Asiatic  countries,  including  the 
Philippine  Islands,  where  it  was  found  by  Strong.  A  few  cases  have 
also  been  encountered  in  the  United  States.  The  pathologic  changes 
present  themselves  as  hypertrophies  of  the  subcutaneous  connective 
tissue,  particularly  along  the  lymphatics.  The  tissue  increase  leads 
to  the  formation  of  distinct  nodules.  If  these  are  incised  they  generally 
discharge  a  thick  pus  or  coagulated  lymph,  which,  under  the  micro- 
scope, shows  yeast-cell-like  bodies  both  within  and  without  the  tissue 
cells.  The  lymph  glands  in  the  neighborhood  of  the  nodules  are 
swollen;  they  sometimes  contain  purulent  foci  which  show  the  para- 
sites. The  latter  may  also  form  metastatic  foci  in  the  internal  organs. 
According  to  Tokishige  the  disease  has  also  been  observed  in  cattle 
in  Japan. 

Morphology. — The  parasite  found  in  the  purulent  and  necrotic 
foci  was  first  seen  by  Rivolta  and  named  Cryptococcus  farciminosus. 
As  later  observations  have  shown  it  belongs  to  the  budding  fungi. 
The  organisms  show  a  double  contoured  membrane;  they  are  round 
or  oval,  and  measure  from  2  to  4  micra  in  length  and  2.5  to  3.6  micra 
in  width.  The  poles  of  the  oval  bodies  are  generally  somewhat  pointed, 
sometimes  buds  are  seen  on  the  cells  which  are  found  in  the  pathologic 
product.  The  contents  of  the  parasitic  cells  are  sometimes  perfectly 
homogeneous;  at  other  times  they  show  a  small  coccus-like  nucleus 
(0.5  to  1  micron  in  diameter),  or  contain  in  their  interior  rather 
coarse  protoplasmic  granules.  Tissue  cells  often  are  full  of  the 
parasites,  and  a  number  of  them  may  also  be  seen  in  leukocytes. 
The  pus  also  contains  shrunken,  irregular,  or  crescentic  blastomyces. 
The  organisms  stain  with  the  ordinary  watery  anilin  stains,  and  are 
Gram  positive. 

Cultural  Properties. — Tokishige  and  others  have  obtained  pure 
cultures  of  these  blastomyces.  They  grow  in  agar,  gelatin,  bouillon, 
on  potatoes,  and  in  other  media,  but  the  development  is  very  slow.  The 
colonies  may  be  visible  after  ten  days,  or  it  may  take  thirty  days 
before  they  appear.  On  agar  they  form  grayish-white  elevated 
granules  from  1  to  4  mm.  in  diameter,  which  become  somewhat  con- 
fluent, and  form  worm-like  or  intestine-like  conglomerations.  The 


CULTURAL  PROPERTIES 


429 


growth  is  comparatively  compact  and  difficult  to  remove  with  the 
platinum  loop.  On  gelatin,  after  several  weeks,  a  yellowish,  sand-like 
mass  is  formed,  liquefaction  of  the  medium  does  not  occur.  On 
potatoes  the  growth  appears  more  rapidly,  and  is  of  a  dirty  white, 
smooth,  lusterless  character.  In  bouillon,  whitish  flocculi,  which 
slowly  sink  to  the  bottom,  are  formed.  Inoculation  of  pure  cultures 
into  horses  and  small  laboratory  animals  is  rarely  if  ever  followed  by 
the  production  of  typical  lesions, 

but  pus  containing   the   organ-  FlG-  175 

isms  when  inoculated  into 
equines  has  produced  the  pic- 
ture of  the  disease.  Tokishige 
was  able  to  infect  horses  and 
to  produce  abscesses  and  nod- 
ules. None  of  the  experi- 
mental inoculations  brought 
about  a  progressive  fatal  case. 
Strong  produced  nodules  in 
monkeys  inoculated  with  pus 


FIG.  174 


Photomicrograph  of  pus  from  a  lymph 
nodule  in  a  horse  suffering  from  blastomy- 
cosis,  showing  intra-  and  extra-cellular  blas- 
tomyces.  (Strong.) 


Blastomycotic  lymphangitis  in  a  North 
Dakota  mare.     (Mohler.1) 


from  naturally  infected  horses.  As  the  organism  in  artificial  cultures 
does  not  ferment  any  of  the  sugars,  the  names  Saccharomyces  farcimi- 
nosus  and  Lymphangitis  saccharomycotica  are  misnomers  and  should 
be  replaced  by  Blastomyces  farciminosus  and  Lymphangitis  blasto- 
mycotica.  The  organism  in  artificial  cultures  forms  mycelia-like 
filaments  composed  of  oblong  or  cylindrical  cells. 


430  BLASTOMYCOTIC  DERMATITIS  IN  MAN 


A  TORULA  AS  THE  CAUSE  OF  A  TUMOR  IN  A  HORSE. 

Frothingham  has  described  a  tumor-like  lesion  in  the  lung  of  a 
horse  caused  by  a  blastomyces.  It  was  situated  in  the  posterior 
portion  of  the  caudal  lobe  of  the  right  lung,  and  was  about  again  as 
large  as  a  human  head.  In  smears  and  sections  a  great  number  of 
blastomycetes  were  seen.  These  were  obtained  in  pure  cultures  on 
potatoes  and  other  media.  On  gelatin  plates  the  organism  formed,  in 
five  to  seven  days,  white,  elevated,  pinhead  colonies.  On  potatoes 
the  growth  was  at  first  white,  soon  becoming  a  dirty  gray,  and  after 
a  few  days  gradually  taking  on  a  chocolate-brown  color.  In  old 
cultures  the  growth  upon  the  less  nutritive  portions  of  the  medium 
becomes  white  and  dry,  and  resembles  lime  deposits.  The  color 
varies  quite  widely;  sometimes  it  remains  a  lighter  or  darker  yellow, 
at  other  times  it  assumes  the  deep  brown  color  almost  immediately. 
The  organisms  are  slightly  oval  and  vary  greatly  in  size.  The  young 
forms  are  surrounded  by  a  delicate  membrane  which  in  older  forms 
becomes  much  thicker.  The  cells  in  cultures  are  sometimes  included 
in  a  gelatinous  matrix.  As  the  organism  during  a  long  observation, 
did  not  produce  spores  nor  ferment  dextrose,  lactose,  or  saccharose, 
it  was  classified  as  a  torula. 

FIG.   176 


Section  through  the  skin  in  a  case  of  blastomycotic  dermatitis  in  man,  showing  small  abscess 
cavity  in  the  epithelial  layers,  near  the  centre  a  budding  parasite.  X  1000.  (Author's  prepar- 
ation.) 

BLASTOMYCOTIC  DERMATITIS  IN  MAN. 

A  disease  of  man,  particularly  observed  in  the  United  States,  and 
known  as  blastomycotic  dermatitis  or  cutaneous  blastomycosis,  is 
due  to  an  organism  or  a  variety  of  organisms  of  the  type  under  dis- 
cussion. Cases  of  this  kind  have  been  reported  by  Gilchrist,  Hektoen, 


BLASTOMYCOTIC  DERMATITIS  IN  MAN 


431 


LeCount,  Montgomery  and  Hyde,  Ricketts,  Anthony  and  Herzog, 
and  others.  Clinically  they  closely  resemble  certain  skin  cancers  or 
the  warty  form  of  skin  tuberculosis  (tuberculosis  verrucosa  cutis),  and 
they  were  formerly  mistaken  for  these  affections.  The  histologic 
features  of  blastomycotic  dermatitis  are  a  hypertrophy  of  the  epithelial 
layers  with  the  formation  of  pegs  and  bands,  as  seen  in  carcinoma, 
and  an  inflammatory  reaction  in  the  derma  and  the  subcutaneous 

FIG.  177 


'A 


Blastomycosis  of  the  skin.     Vertical  section  from  a  typical  lesion,     a,  hyperplasia  of  rete; 
6,  abscesses  in  epithelium;  c,  infiltration  of  cutis.      X  55. 

connective  tissue.  In  the  hypertrophied  masses  of  epithelial  cells 
miliary  abscesses  are  found  which  contain  the  budding  fungi.  These 
can  best  be  demonstrated  in  sections  by  the  eosin-methylene-blue  stain, 
and  they  can  also  be  seen  in  squeezed-out  pus  in  the  unstained,  moist 
cover-glass  preparation.  Hyde  and  Montgomery  have  described 
pure  cultures  of  these  organisms  raised  on  glycerin  and  glucose  agar 
as  follows: 
The  time  required  for  the  development  of  the  different  organisms 


432 


BLASTOMYCOTIC  DERMATITIS  IN  MAN 


in  the  original  cultures  varies  from  two  to  sixteen  days,  the  majority 
showing  a  growth  in  from  two  to  eight  days.  Subcultures  appear  in 
from  two  to  five  days.  In  gross  appearances  the  cultures  may  show 
slightly  elevated,  white,  smooth  colonies  or  irregular  areas  following 
the  track  of  the  needle;. a  translucent,  gelatinous  or  yellowish-brown 
and  pasty  growth;  a  roughly  granular  surface,  which  may  eventually 
form  prominent  folds  and  depressions;  a  light,  white  (in  older  cultures 
slightly  yellow  or  yellowish  brown)  fluffy  growth,  with  short  or  long 
aerial  hyphae;  or  a  central  white^  elevated  portion,  which  may  be 
fluffy  or  covered  with  short  projections  like  white  hairs,  and  which 
is  surrounded  by  a  translucent,  non-elevated  zone.  With  very  few 
exceptions,  the  growth  extends  more  or  less  into  the  medium  and 
becomes  closely  incorporated  with  it. 


FIG.  178 


FIG.  179 


Blastomycosis  of  the  skin.     Budding 
organism  in  tissue.     X  1200. 


Blastomycosis  of  the  skin.     Hanging 
drop.      X  1200. 


Moist  preparations  from  the  cultures  may  show  budding  organisms; 
or  fine,  homogeneous,  and  branching  mycelia,  more  or  less  segmented, 
with  or  without  lateral  conidia,  which  may  contain  few  or  many 
highly  refractive  bodies,  varying  in  size.  The  latter  are  probably 
spores,  though  in  some  instances  they  may  be  oil  drops.  Mingled 
with  the  mycelium  may  be  seen  round,  oval,  or  irregular  double- 
contoured  bodies,  varying  greatly  in  size,  and  more  or  less  filled  with 
highly  refractive  globular  bodies. 

The  organism  in  a  number  of  the  reported  cases  has  formed  mul- 
tiple metastatic  abscesses  through  the  general  circulation  and  lead  to 
death.  The  blastomycotic  infection  in  man  as  the  similar  infection 
in  the  horse  in  pseudofarcy  is,  therefore,  a  dangerous  disease. 


QUESTIONS  433 


BLASTOMYCES  THE  CAUSE  OF  TUMORS? 

A  number  of  investigators,  particularly  Sanfelice,  have  claimed 
that  blastomyces  are  the  cause  of  malignant  tumors  in  man  and  the 
lower  animals,  but  these  claims  have  not  been  confirmed.  Sanfelice 
reported  the  finding  of  a  pathogenic  blastomyces  in  a  cancer  of  the 
liver  in  an  ox,  and  since  the  pathologic  lesion  was  calcified  he  named 
the  organism  Saccharomyces  lithogenes  (lithogenes,  stone-forming). 
Another  organism  obtained  from  nodules  in  the  lung  of  a  hog  was 
described  by  the  same  author  as  Saccharomyces  granulomatogenes.1 


QUESTIONS. 

1.  What  is  the  common  English  name  for  Saccharomyces? 

2.  What  is  the  meaning  of  the  term  blastomyces? 

3.  Describe  the  process  of  budding. 

4.  Why  have  saccharomycetes  been  classified  as  ascomycetes? 

5.  Define  the  terms:  ASCIIS,  endogenous  ascospares. 

6.  What  is  the  difference  between  a  typical  Saccharomyces  and  a  torula? 

7.  Do  saccharomycetes,  by  budding,  always  form  new  individual  unicellular 
globular  organisms?     If  not,  what  do  they  form? 

8.  Describe  the  finer  details  of  the  structure  of  a  blastomyces. 

9.  How  can  blastomyces  be  stained  in  cover-glass  preparations?     How  in 
tissue  sections? 

10.  What  is  pseudofarcy  in  horses? 

11.  Describe  its  pathologic  lesions. 

12.  Name  and  describe  its  cause. 

13.  Describe  the  cultural  properties  of  the  Blastomyces  farciminosus. 

14.  Have  any  other  blastomyces  been  found  in  pathologic  lesions  in  horses? 

15.  What  is  blastomycetic  dermatitis  in  man? 

16.  Describe  its  pathologic  lesions. 

17.  Describe  the  organism  causing  it. 

18.  What  is  Saccharomyces  lithogenes  and  Saccharomyces  granulomatogenes? 

1  A  complete  list  of  budding  fungi  occasionally  found  in  pathologic  lesions  is  given  in  Busse'a 
contribution  on  "Sprosspilze"  in  Kolle  and  Wassermann's  Manual,  vol.  i,  p.  661. 


28 


CHAPTER    XL. 

BACTERIA,  GENERALLY  NOT  PATHOGENIC,  OFTEN  EMPLOYED  IN 
LABORATORY  PRACTICE— BACILLI  OF  THE  PROTEUS  GROUP- 
BACILLUS  ANTHRACOIDES— BACILLUS  MEGATHERIUM- 
BACILLUS  PRODIGIOSUS— BACILLUS  VIOLACEUS 
—BACILLUS  CYANOGENUS— MICROCOCCUS 
TETRAGENUS— MICROCOCCUS  AGILIS 
— SARCINA  LUTEA. 

Bacilli  of  the  Proteus  Group. — Members  of  this  group  were  first 
isolated  from  decaying  animal  material.  They  are  aerobic  and 
facultative  anaerobic  organisms  which  in  their  growth  decompose 
proteid  materials  and  produce  a  very  fetid  smell.  They  are  named 
proteus  because  they  are  exceedingly  variable  in  their  morphology. 
While  in  their  most  typical  shape  they  are  bacilli  of  medium  size,  they 
appear,  particularly  in  older  cultures,  in  short  coccoid  form  and  also 
in  curved  vibrio-like  shape.  They  stain  with  ordinary  watery  anilin 
solutions,  but  are  generally  Gram  negative  and  do  not  form  spores. 
The  most  common  type  of  this  group  is  the  Bacillus  proteus  vulgaris 
of  Hauser.  This  organism  varies  in  length  from  1.2  to  4  micra  and 
more  and  is  0.6  micron  wide.  It  is  lively  motile  and  possesses  a  large 
number  of  flagella  distributed  around  the  entire  body.  The  growth 
on  5  per  cent,  gelatin  is  very  typical.  At  room  temperature  round, 
depressed,  whitish  colonies  are  formed  which  send  out  wreaths  of 
filamentous  projections  into  the  surrounding,  not  yet  liquefied,  medium. 
As  the  liquefaction  goes  on  the  peripheral  portions  of  the  growth 
break  away  from  the  principal  mass  and  swarm  about  in  the  liquefied 
medium.  This  wandering  away  can  be  observed  under  a  .medium  or 
low  power  of  the  microscope.  In  firmer  gelatin  of  a  higher  concentra- 
tion this  swarming  of  broken-off  portions  of  the  colonies  does  not 
occur.  The  growth  is  best  at  24°  C.,  but  it  is  still  quite  abundant  at 
37°  C.  The  bacillus,  when  grown  under  anaerobic  conditions,  does 
not  liquefy  gelatin.  Upon  agar  the  bacillus  forms  a  moist,  thin, 
transparent  growth.  Milk  is  coagulated.  It  produces  indol  and 
phenol  and  reduces  nitrates  to  nitrites.  Glucose  and  saccharose  are 
fermented,  but  not  lactose.  If  injected  in  small  amount  subcutaneously 
into  animals  no  ill  effect  is  produced.  Larger  doses  introduced 
intravenously  or  intraperitoneally  produce  death  under  symptoms 
pointing  to  intoxication.  The  Bacillus  proteus  vulgaris  does  not 
multiply  in  healthy  tissues,  but  it  can  grow  in  necrotic  tissues,  and 
has  been  found  in  wounds.  It  furnishes  a  soluble  toxin  which  causes 


BACILLUS  MEGATHERIUM 


435 


the  intoxication  in  intravenous  or  intraperitoneal  injection.  The 
filtrate  also  contains  a  hemotoxin  which  dissolves  red  blood  corpuscles. 
Meat  infected  with  the  organism  has  been  known  to  cause  gastro- 
intestinal disturbances.  The  bacillus  if  injected  into  the  bladder 
of  an  animal  causes  cystitis;  it  has  also  been  found  in  man  as  the 
cause  of  cystitis.  The  Bacillus  proteus  mirabilis  is  a  variety  of  the 
Bacillus  proteus  vulgaris ;  it  liquefies  gelatin  more  slowly,  but  otherwise 
closely  resembles  the  proteus.  The  Bacillus  proteus  Zenkeri,  another 
variety,  does  not  liquefy  gelatin.  The  Bacillus  proteus  Zopfii,  or 
Bacterium  Zopfii,  has  been  isolated  from  the  intestines  of  chickens. 


FIG.  180 


FIG.  181 


Bacillus  proteus  vulgaris.      X  1000. 
(Author's   preparation.) 


Bacillus  anthracoides,  beginning  spore  forma- 
tion.     X  1000.     (Author's  preparation.) 


Bacillus  Anthracoides. — This  organism  is  found  in  water  and  soil. 
Morphologically  and  in  cultures  it  very  much  resembles  the  Bacillus 
anthracis,  but  it  is  no  wise  pathogenic.  Its  ends  are  a  little  more 
rounded  than  those  of  the  bacillus  of  anthrax;  its  growth  on  the 
laboratory  culture  media  is  like  that  of  true  anthrax.  Spore  formation, 
however, 'is  better  at  room  than  at  incubator  temperature.  Mice  and 
guinea-pigs  can  be  inoculated  without  any  evil  effects.  A  Bacillus 
pseudoanthracis  has  been  described  by  Burri.  It  is  somewhat  like 
the  anthrax  bacillus,  but  motile,  non-pathogenic,  and  differing  mark- 
edly in  its  cultures. 

Bacillus  Megatherium. — This  organism  is  found  on  plants  and  in 
the  soil  and  air.  It  is  named  megatherium1  because  it  is  a  very  large, 
plump,  and  very  sluggishly  motile  bacillus.  It  measures  10  micra  and 
more  in  length  and  is  2.5  micra  thick,  and  generally  occurs  in  short 
chains.  Short  involution  forms  are  frequently  formed  in  older  cultures 
or  on  unfavorable  media.  The  protoplasm  of  the  bacillus  frequently 
appears  finely  granular.  It  forms  spores,  and  these  leave  the  bacillus, 

1  Megatherium  is  the  fossil  giant  sloth  of  South  America. 


436     BACTERIA  OFTEN  EMPLOYED  IN  LABORATORY  PRACTICE 

not  at  either  end,  but  in  the  equatorial  plane.  The  bacillus  mega- 
therium is  strictly  aerobic;  on  gelatin  the  colonies  are  kidney-shaped 
or  crescentic  and  granular  after  a  few  days'  growth.  The  medium 
is  liquefied  in  stick  cultures  in  a  funnel-shaped  manner.  On  agar  a 
whitish  film  is  developed  and  on  potatoes,  a  thick,  smeary,  grayish- 
white  or  yellowish  layer. 

Bacillus  Prodigiosus. — This  "wonderful'1  bacillus,  so  called  on 
account  of  the  beautiful  red  pigment  which  it  forms,  is  a  small  rod, 
0.5  to  1  micron  long,  and  was  formerly  mistaken  for  a  coccus.  The 
rod  shape  is  most  marked  in  slightly  acid  media  (best  acidulated 
with  tartaric  acid  or  boric  acid).  The  bacillus  is  motile,  and  possesses 
flagella,  arranged  on  one  side.  It  does  not  form  spores,  but  can 
resist  drying  out  for  a  considerable  time;  it  often  forms  yeast-like 
involution  forms.  Pigment  formation  is  best  at  20°  to  24°  C.  Gelatin 
is  liquefied  rapidly.  On  agar  the  colonies  are  at  first  without  color, 
but  it  appears  later.  The  pigment  is  only  formed  in  the  presence 
of  oxygen.  The  most  intense  color  is  formed  on  potatoes.  Milk  is 
likewise  stained  red.  Sugar  is  fermented  by  this  organism. 

A  considerable  number  of  different  bacilli  occurring  in  water  form 
a  red  pigment,  as  the  Bacillus  ruber  aquatilis,  Bacillus  rubefaciens, 
Bacillus  rubescens,  etc. 

Bacillus  Violaceus. — This  organism  has  frequently  been  found  in 
water.  It  is  a  motile  bacillus  0.8  by  1.7  micron;  it  generally  occurs 
in  pairs,  forms  oval  spores,  and  grows  at  room  temperature,  but  not 
at  37°  C.  In  gelatin  the  colonies  first  appear  like  small  air-bubbles 
in  the  medium.  They  are  irregular  in  outline  and  rapidly  liquefy 
the  culture  soil.  In  stick  cultures  the  liquefaction  leads  to  the  for- 
mation of  a  funnel,  at  the  bottom  of  which  is  a  violet  sediment.  On 
potatoes  the  pigment  formed  is  of  a  dark,  black  violet;  on  agar  of  a 
bright,  lacquer-like  violet  color.  Blood  serum  is  also  liquefied; 
nitrates  are  reduced  to  nitrites.  The  pigment  of  this  bacillus  under- 
goes various  changes  when  treated  with  a  variety  of  dilute  acids. 
Mineral  acids  change  the  violet  to  blue  green,  chlorine  water  to 
yellow,  hydrate  of  sodium  solution  to  brownish  yellow,  ammonia  to 
bluish  green.  The  ordinary  Bacillus  violaceus  and  a  number  of  its 
varieties  described  are  absolutely  non-pathogenic.  Wooley,  however, 
encountered  a  Bacillus  violaceus  (var.  Manilse)  in  the  Philippine 
Islands  which  appears  to  have  caused  the  death  of  several  water 
buffaloes,  and  which,  after  being  obtained  in  pure  cultures,  was  very 
pathogenic  for  rabbits. 

Bacillus  Cyanogenus. — This  organism  causes  the  blue  discoloration 
of  milk.  It  has  been  known  for  a  long  time,  but  was  not  obtained  in 
pure  cultures  until  isolated  by  Hiippe.  It  varies  in  size  from  1  to  4 
micra  by  0.3  to  0.5  micron  in  thickness.  It  is  motile  and  possesses  a 
number  of  flagella  at  one  end.  It  is  Gram  negative  and  grows  best  at 
room  temperature,  not  at  37°  C.  It  is  strictly  aerobic.  In  milk  it  does 
not  form  acid,  but  alkali,  and  does  not  coagulate  it.  The  organism 


SARCINA  437 

forms  a  fluorescent  pigment  and  a  non-fluorescent-blue  to  blue-black 
pigment.  The  blue  character  of  the  latter  is  best  shown  in  an  acid 
medium;  it  is  black  when  the  reaction  is  neutral,  brown  when 
the  reaction  is  alkaline.  A  rose-red  color  frequently  precedes  the 
blue  pigmentation.  The  blue  discoloration  appears  in  milk  only 
when  it  is  acid,  and  is  for  this  reason  best  produced  after  some 
development  of  lactic-acid  bacteria.  The  bacillus  is  absolutely 
non-pathogenic. 

Micrococcus  Tetragenus. — This  organism  is  named  from  the  fact 
that  it  is  generally  found  in  tetrads,  i.  e.,  groups  of  four.  The  indi- 
vidual cocci  are  about  1  micron  in  diameter.  The  organism  is  fre- 
quently found  in  sputum  and  discharges  from  the  nose.  Under  these 
conditions  it  possesses  a  gelatinous  envelope  which  generally  surrounds 
the  group  of  four.  In  cultures  the  cocci  are  seen  singly,  ill  pairs  and 
as  tetrads.  The  coccus  stains  easily  with  the  watery  anil  in  solutions 
and  keeps  Gram's  stain.  On  gelatin  the  organism  first  forms  small, 
whitish  points  which  later  develop  into  thick,  elevated,  moist  drops 
of  1  to  2  mm.  in  diameter.  They  become  confluent  and  finally  form 
a  continuous  moist  growth.  Gelatin  and  blood  serum  are  not  liquefied. 
On  agar  the  growth  is  not  as  abundant  as  on  gelatin,  but  on  potatoes 
it  is  very  luxuriant.  The  organism  is  pathogenic  to  white  mice.  If 
small  doses  are  injected  these  animals  become  somnolent  and  quiet 
after  two  days,  and  they  die  three  to  six  days  after  the  injection. 
The  Micrococcus  tetragenus  is  then  found  in  large  numbers  in  the 
internal  organs  of  the  dead  mice.  Gray  house  mice  are  immune; 
guinea-pigs  develop  local  abscesses  or  a  general  septicemia.  Intra- 
peritoneal  injection  leafls  to  a  purulent  peritonitis.  Larger  animals, 
such  as  rabbits,  dogs,  etc.,  are  not  susceptible. 

Micrococcus  Agilis. — This  organism  was  first  isolated  from  drinking 
water.  It  is  one  of  the  few  cocci  which  possess  a  flagellum,  in  con- 
sequence of  which  they  are  motile.  The  coccus  has  a  diameter  of 
about  1  micron;  it  grows  on  the  ordinary  media  at  room  temperature 
and  forms  a  rose-red  pigment.  Gelatin  becomes  liquefied.  The 
organism  forms  pairs,  tetrads,  or  short  chains.  Its  motility  can  best 
be  exhibited  in  media  containing  5  per  cent,  lactose.  It  can  be 
demonstrated  that  every  coccus  possesses  one  flagellum.  When  several 
cocci  adhere,  particularly  in  tetrad  form,  the  group  generally  performs 
a  kind  of  rotary  motion  around  its  own  axis.  Loeffler  and  Menge  have 
described  a  motile  coccus  forming  a  yellow  pigment  under  the  name 
of  Micrococcus  agilis  citreus. 

Sarcinae. — All  sarcinae  are  cocci  which  in  multiplication  arrange 
themselves  in  square  groups  or  packages  which  have  been  likened 
to  a  bale  of  cotton.  Sarcina  lutea  is  composed  of  comparatively  large 
cocci  about  1  micron  or  more  in  diameter.  The  sarcina  form  cannot 
be  as  readily  seen  in  stained  specimens  as  in  the  hanging  drop.  The 
growth  of  the  organism  on  gelatin  is  at  first  slow  and  leads  to  the 
formation  of  point-like  colonies  which  later  become  larger  and 


438     BACTERIA  OFTEN  EMPLOYED  IN  LABORATORY  PRACTICE 

irregular  in  outlines  and  flow  together  forming  a  thick,  moist,  lemon- 
colored  growth. 

Sarcina  aurantiaca  forms  an  orange-yellow  pigment;  Sarcina  alba 
a  white,  and  Sarcina  rubra  a  red  pigment.  Sarcina  mobilis  is  remark- 
able for  the  fact  that  it  is  motile  and,  as  claimed,  possesses  a  flagellum. 
Sarcina  ventriculi  is  found  in  the  gastric  contents  of  man  and  animals. 
The  individual  cocci  are  very  large  (up  to  2.5  micra  in  diameter)  and 
they  are  found  in  groups  of  eight.  Whether  Sarcina  ventriculi  is  a 
definite  species  or  not,  or  simply  represents  varieties  of  sarcinae  taken 
up  with  food  or  water,  is  a  question  not  yet  settled. 


QUESTIONS. 

1.  What  is  the  relation  of  members  of  the  proteus  group  of  bacilli  to  proteid 
material? 

2.  Why  have  these  bacilli  received  the  name  proteus? 

3.  Describe  the  variability  of  their  shape. 

4.  Describe  the  morphology  and  the  cultural  properties  of  Bacillus  proteus 
vulgaris. 

5.  What  is  meant  by  the  swarming  islands  of  a  proteus  gelatin  culture? 

6.  Has  the  Bacillus  proteus  any  pathogenic  properties? 

7.  What  is  the  Bacillus  anthracoides? 

8.  -What  is  a  megatherium? 

9.  Describe  the  Bacillus  megatherium  and  state  why  it  has  received  this 
peculiar  name. 

10.  Describe  the  Bacillus  prodigiosus.     Why  so  named? 

11.  Are  there  other  chromogenic  bacilli  which  produce  a  red  pigment? 

12.  Describe  the  Bacillus  violaceus.     Is  it  ever  pathogenic? 

13.  Describe  the  Bacillus  cyanogenus. 

14.  Describe  the  Micrococcus  tetragenus.     What  does  tetragenus  mean? 

15.  Is  this  micrococcus  pathogenic  for  any  animal? 

16.  What  unusual  feature  does  the  Micrococcus  agilis  possess? 

17.  What  is  a  sarcina  in  general?    Describe  the  Sarcina  lutea. 

18.  Describe  the  Sarcina  aurantiaca. 

19.  Where  is  Sarcina  ventriculi  found? 


CHAPTEE    XLL 

INFECTIOUS  DISEASES  DUE  TO  ULTRAMICROSCOPIC  VIRUSES— 

PLEUROPNEUMONIA  IN  CATTLE— CATTLE  PLAGUE— 

HOOF-AND-MOUTH  DISEASE. 

IT  would  be  illogical  to  assume  the  non-existence  of  bacteria  so 
small  that  they  cannot  be  recognized,  even  with  the  best  microscopic 
objectives  and  oculars.  In  fact,  at  least  one  pathogenic  micro- 
organism of  this  type  is  already  known,  though  even  under  the 
best  optical  apparatuses  it  appears  only  as  a  small,  highly  refractive 
point.  Yet  from  its  behavior  there  is  every  reason  to  believe  that 
it  is  indeed  a  bacterium.  The  organism  referred  to  is  the  exceedingly 
minute  microorganism  discovered  by  Nocard  and  Roux  as  the  cause 
of  pleuropneumonia  in  cattle.  In  albuminous  natural  exudates  it 
will  not  pass  the  Chamberland  or  Berkefeld  filters,  but  it  will  pass 
these  if  suspended  in  watery  solutions,  such  as  bouillon,  etc.  It  can 
be  cultivated  on  certain  artificial  media  and  forms  something  like 
very  delicate,  hardly  visible  colonies.  A  further  step  in  reasoning 
leads  to  a  living  virus  which  may  or  may  not  pass  the  filters  in 
albuminous  exudates,  and  which  cannot  be  seen  even  as  a  highly 
refractive  point.  Another  step  leads  to  a  living  virus  so  small 
that  it  easily  passes  the  pores  of  porcelain  and  clay  filters  even  in 
natural  albuminous  fluids.  With  this  the  form  of  "contagium 
vivum  fluidum"  is  reached  which  is  designated  as  an  ultramicroscopic, 
invisible,  filterable,  living  virus.  Some  of  the  diseases  due  to  such 
viruses  which  are  either  still  on  the  boundary  line  of  visibility  or 
which  are  absolutely  invisible  and  perhaps  never  can  be  seen  are 
briefly  considered  in  this  chapter.1 


CONTAGIOUS  PLEUROPNEUMONIA  IN  CATTLE. 

Occurrence  and  Historical. — Lung  plague  in  cattle,  pleuropneumonia 
contagiosa  bovum,  "Lungenseuche  der  Kinder"  (German),  peri- 
pneumonie  contagieuse  (French),  is  an  acute  or  subacute  infectious 
disease  of  cattle  characterized  by  exudative  inflammatory  changes 
in  the  lungs  combined  with  sero-fibrinous  pleuritis.  The  disease 

1  Helmholtz,  the  celebrated  German  physicist,  has  shown  that  in  consequence  of  the  inherent 
properties  of  light,  it  will  be  impossible,  no  matter  how  much  the  microscope  is  improved,  to 
see  objects  smaller  than  a  certain  minimum  which  he  calculated  exactly.  There  is  of  course, 
no  reason  whatever  for  assuming  that  there  are  no  live  objects  in  nature  beyond  this  micro- 
scopic limit. 


440  CONTAGIOUS  PLEUROPNEUMONIA  IN  CATTLE 

is  caused  by  the  smallest  known  bacterium,  which  was  discovered 
by  Nocard  and  Roux  in  1898.  The  epizootic  was  first  correctly 
described  by  Bourgelat  in  France  (1765),  but  it  had  previously  been 
noticed  in  Germany.  It  has  prevailed  throughout  Europe  and  was 
imported  to  Africa,  Asia,  and  Australia,  and  introduced  into  the 
United  States,  probably  first  into  New  York  in  1843.  It  then  spread 
to  various  States,  but  it  was  vigorously  opposed  and  finally  stamped 
out  in  1891,  since  which  time  this  country  has  been  free  from  the 
disease. 

Pathologic  Lesions. — The  principal  lesions  are  generally  found 
only  on  one  side  of  the  thorax  and  on  the  pleura  of  the  same  side, 
but  there  appears  to  be  no  predilection  as  to  the  side  involved.  This 
is  true  of  75  per  cent,  of  the  animals  affected.  According  to  Nocard, 
the  pleura  is  always  involved,  but  in  very  variable  degrees.  Sometimes 
when  the  inflammatory  changes  in  the  lung  greatly  predominate  a 
certain  amount  of  thickening  and  infiltration  is  seen  in  the  pleura 
pulmonalis  of  the  same  side.  In  a  more  advanced  stage  there  is 
vascularization  of  the  serous  membrane,  with  an  abundant  fibrinous 
exudate  on  its  surface.  The  lungs  in  acute  cases  are  found  hepatized 
in  more  or  less  extensive  areas;  they  are  void  of  air  and  non-elastic. 
On  section  a  clear,  serous,  yellowish  fluid  oozes  out  of  the  areas  of 
hepatization.  When  collected  in  a  clean  vessel  this  fluid  subsequently 
coagulates  into  a  gelatinous  mass.  The  interlobular  connective 
tissue  is  increased  and  forms  a  light  yellowish  network  which  divides 
the  hepatized  area  into  irregular  patches  of  various  colors.  The 
latter  may  be  gray,  light  red  or  dark  brown  red,  so  that  a  cut  surface 
presents  a  mottled  appearance.  The  increased  interalveolar  con- 
nective tissue  shows  enlarged  lymph  vessels  and  clefts  filled  with  a 
yellowish  serous  or  a  more  fibrinous  exudate.  The  obliterated 
alveoli  of  an  area  of  hepatization  are  sometimes  light  red  and 
firm  at  the  periphery,  while  the  centre  of  the  solid  tissue  has 
become  dark  red  and  soft-elastic.  The  walls  of  the  bronchi  in  the 
affected  pulmonary  areas  are  infiltrated,  their  lumina  contain  an 
exudate  and  the  peribronchial  and  mediastinal  glands  are  quite 
edematous.  While  the  pleura  over  the  diseased  portions  of  lungs 
generally  shows  the  changes  described  above,  it  may  also  contain  very 
little  fluid  between  the  visceral  and  the  parietal  layer  or,  on  the  other 
hand,  a  large  amount  of  clear  yellow  fluid  or  a  yellowish-gray  cloudy 
fluid  with  flocculi  of  fibrin  may  be  present.  When  only  very  small 
amounts  of  pleuritic  exudate  are  present  the  condition  is  known  as 
pleuritis  sicca.  On  the  other  hand  the  amount  of  fluid  may  be  15  to 
20  liters.  In  some  cases  the  greater  portion  of  the  exudate  is  not 
found  free  between  the  two  layers  of  the  pleura,  but  has  infiltrated 
the  mediastinal  connective  tissue.  The  latter  then  forms  a  soft 
gelatinous  tumor  composed  of  confluent,  infiltrated  masses  of  a  yellow 
color.  If  cut  into,  these  masses  discharge  an  abundant  amount  of 
yellowish  or  decidedly  amber-colored  serous  fluid. .  The  description 


BACTERIOLOGY  441 

as  far  as  given  refers  more  particularly  to  the  acute  form  of  the 
disease. 

In  the  more  chronic  form  there  is  considerable  fibrosis  of  the 
affected  lung,  i.  e.,  new  formation  of  inflammatory  connective  tissue. 
Areas  in  the  lung  appearing  almost  as  solid  as  meat  (a  condition 
known  as  carnificatiori)  alternate  with  necrotic  and  occasionally 
calcified  areas.  The  necrotic  portions,  or  sequesters,  may  assume  a 
considerable  size  and  may  bulge  as  nodular  masses  above  the  general 
pleural  surface.  The  sequesters  are  frequently  surrounded  by  a 
tough,  fibrous,  connective  tissue  capsule  and  discharge,  after  an 
incision  through  the  latter,  a  mushy,  dirty  mass  and  more  solid 
particles.  Sometimes  such  softened,  necrotic  portions  of  the  lung 
break  into  a  bronchus  and  are  discharged  through  it  into  the  outside 
world.  They  vary  in  size  from  a  walnut  to  a  child's  head,  and  they 
may  be  formed  after  a  preliminary  purulent  infiltration,  or  the  serous 
yellowish  exudate  may  so  compress  bloodvessels  and  lymphatics 
that  portions  of  the  lung  are  deprived  of  their  nutrition  and  necrosis 
results  from  the  lack  of  blood  supply. 

Histologic  studies  by  Pourcelot  and  MacFadyean  have  shown  that 
the  first  tissue  changes  occur  in  the  interalveolar  septa  and  that  their 
lymphatics  become  distended  with  a  finely  granular  fibrinous  coagulum 
containing  a  few  cells.  The  alveoli  at  this  early  stage  show  no 
marked  changes,  a  little  later,  however,  the  zone  adjacent  to  the 
alveolar  wall  secretes  a  fibrinous  exudate  which  extends  toward  the 
centre.  At  the  same  time  the  interalveolar  connective  tissue  becomes 
infiltrated  with  leukocytes  and  subsequently  thickened  by  newly 
formed  connective  tissue.  The  mucosa  becomes  covered  with  a 
fibrinous  exudate.  The  changes  in  the  pleura  first  consist  in  an 
edematous  infiltration,  later  the  surface  becomes  covered  with  a 
fibrinous  exudate,  false  membranes  are  formed,  and  these  finally 
become  organized  and  replaced  by  cicatricial  connective  tissue. 
The  pericardium  is  sometimes  involved  and  the  peritoneal  cavity 
occasionally  contains  some  fluid  and  an  exudate  upon  its  surface. 

Bacteriology. — Nocard  describes  the  bacteriology  of  the  disease  as 
follows:  The  examination  of  stained  specimens  from  the  virulent 
serous  exudate  infiltrating  the  alveoli  does  not  show  any  bacteria.  This 
serous  fluid,  however,  when  properly  preserved  or  mixed  with  bouillon, 
does  not  lose  its  virulency.  In  spite  of  the  fact  that  it  apparently 
contains  no  microorganisms,  its  inoculation  into  a  place  as  unfavorable 
as  the  end  of  the  tail  of  a  cow  produces  considerable  reaction  and 
sometimes  death.  If  heated  to  from  65°  to  70°  C.  the  exudate  loses 
its  virulency.  This  also  occurs  after  certain  processes  of  filtration. 
If  collodion  sacs,  inoculated  with  a  trace  of  the  exudate  obtained 
under  aseptic  precautions,  are  implanted  into  the  peritoneal  cavities 
of  rabbits  and  re-obtained  after  fifteen  to  twenty  days,  the  contents 
of  the  sacs  have  become  opalescent  and  slightly  cloudy.  Upon 
microscopic  examination  of  the  contents  neither  cells  nor  bacteria 


442  CONTAGIOUS  PLEUROPNEUMONIA  IN  CATTLE 

are  seen  which  could  be  cultivated  upon  the  ordinary  culture  media. 
Examination,  however,  with  very  good  objectives  and  strong  light  at 
a  magnification  of  1500  to  2000  diameters,  show  innumerable  very 
small  motile  and  highly  refractive  points.  These  are  live  organisms 
which,  protected  against  phagocytosis  by  the  collodion  sac,1  have  been 
able  to  multiply. 

They  can  also  be  cultivated  outside  of  the  living  body,  in  a  special 
bouillon  prepared  according  to  Martin,  as  follows:  Peptone  from 
digested  pig's  stomach,  5  parts  to  100  parts  of  cattle  or  rabbit  serum. 
This  must  be  freed  from  bacteria  by  filtration  through  a  Pasteur 
filter,  but  must  not  be  heated.  If  this  medium  is  inoculated  from 
collodion  sacs  and  incubated  at  37°  C.  for  two  or  three  days  it  becomes 
opalescent,  and  if  shaken  there  is  evidence  of  slight  clouding.  The 
highest  magnifications  show  exceedingly  minute  highly  refractive 
granules,  so  small  that  no  conclusions  as  to  their  shape  can  be  drawn. 
These  cultures  are,  however,  virulent  and  they  can  also  be  used  for 
immunizing  inoculations.  Solid  culture  media  can  be  prepared  by 
adding  sterile  melted  gelatin  to  Martin's  liquid  medium.  If  a  drop 
of  pulmonary  exudate  or  of  fluid  culture  is  allowed  to  run  over  the 
surface  of  the  gelatin  a  large  number  of  very  delicate  transparent 
colonies  can  be  seen  after  three  or  four  days  with  the  aid  of  a  micros- 
cope. A  little  later  the  colonies  become  denser  and  opaque  in  the  centre. 
They  finally  cover  the  surface  with  a  veil  which  adheres  so  firmly  that 
it  can  only  be  removed  by  breaking  up  the  culture  medium.  If  the 
albuminous  exudate  or  a  culture  in  Martin's  liquid  medium  is  filtered 
through  a  Chamberland  or  Berkefeld  filter  a  sterile  fluid  is  obtained; 
if,  however,  the  fluids  are  diluted  with  a  watery  liquid  the  microbes 
pass  through.  The  latter  fluid  is  fully  virulent.  The  organism  is 
aerobic;  it  does  not  multiply  in  vacua  or  in  inert  gases;  the  growth  is 
always  slow  and  scanty,  and  its  optimum  temperature  is  at  36°  to  38°  C. 
The  microbe  takes  the  basic  aniline  stains,  but  it  is  so  small  that 
even  after  prolonged  staining  with  strong  solutions  the  shape  cannot 
be  ascertained  and  the  fine  colored  dusty  particles  which  are  seen 
are  apparently  each  composed  of  numerous  individual  microbes.  If 
impression  preparations  are  made  the  mass  on  the  cover-glass  takes 
the  stain.  It  is  decolorized  if  treated  by  Gram's  method. 

Animals  Susceptible. — Cattle,  buffaloes,  yak,  and  bison  are  the  only 
susceptible  animals.  Natural  infection  occurs  by  close  contact 
between  cattle.  While  there  is  some  evidence  that  infection  occurs 


1  The  fluid  to  be  inoculated  into  collodion  sacs  is  obtained  in  the  following  manner  (Nocard): 
It  is  necessary  to  procure  a  lung  from  an  animal  in  an  early  stage  of  the  acute  form  of  the 
disease.  A  place  is  selected  which  indicates  a  consolidation  and  a  collection  of  fluid  behind 
the  pleura.  The  latter  is  then  superficially  cauterized  with  a  hot  knife  or  steel  spatula.  A  fine 
pointed,  sterile,  glass  pipette  is  pushed  through  the  cauterized  pleura  into  the  layers  of  the 
tissue  next  to  it,  and  about  0.5  c.c.  of  the  clear  amber-colored  fluid  may  be  so  obtained.  From 
this  collodion  sacs  containing  sterile  bouillon  are  inoculated,  and  these  after  having  been 
sealed  at  the  glass  end  are  implanted  into  the  peritoneal  cavity  of  rabbits.  Martin's  medium 
may  also  be  inoculated  from  the  amber  fluid  obtained  by  puncture  of  the  pleura. 


PATHOLOGIC  LESIONS  443 

by  inhalation,  experiments  to  spread  the  disease  by  this  method  have 
generally  been  unsuccessful.  The  virus  may  remain  alive  and  active 
for  some  time  in  stables  where  sick  animals  have  been  kept  and  it  may 
spread  to  new  importations  even  after  the  disease  has  disappeared 
among  the  stock  by  deaths  or  recoveries.  If  fluid  containing  the 
microorganism  of  the  disease  is  inoculated  in  a  very  small  dose 
into  the  subcutaneous  connective  tissue  of  cattle  an  edematous, 
painful  swelling  appears  after  a  variable  number  of  days  at  the 
point  of  inoculation,  the  temperature  rises  to  41°  to  42°  C.,  and 
death  occurs  after  a  fall  of  temperature  and  a  comatose  condition 
have  set  in. 

Protective  Inoculation. — This  has  been  practised  for  many  years, 
first  in  an  empirical  manner,  and  in  1852  by  Willems,  who  dipped 
a  vaccination  lancet  into  the  juice  expressed  from  the  alveoli  of  an 
infected  lung,  and  vaccinated  in  two  or  three  places  at  the  lower  end 
of  the  tail.  Pasteur,  in  1881  and  1882,  used  a  lymph  attenuated  by 
mixing  the  fluid  from  affected  lungs  with  glycerin.  A  large  number 
of  investigators  have  at  various  times  attempted  to  devise  an  effective 
protective  inoculation.  Raebiger,  however,  who  has  critically 
reviewed  the  various  methods,  comes  to  the  conclusion  that  their 
protective  value  is  problematical,  that  the  danger  of  contraction  of 
the  disease  by  non-immune  animals  from  vaccinated  animals  is  great, 
and  that  the  best  method  of  limiting  the  disease  consists  in  the  slaughter 
of  all  infected  animals.  If  for  any  reason  this  is  impossible,  pro- 
tective vaccinations  by  attenuated  lymph  or  artificial  pure  cultures 
may  be  practised. 

CATTLE  PLAGUE. 

Occurrence. — Cattle  plague,  contagious  typhus,  pestis  bovina,  or 
Rinderpest  (German),  is  a  highly  contagious,  very  fatal  (60  to  90  per 
cent,  mortality)  disease  of  cattle.  It  is  very  prevalent  in  various  parts 
of  Asia,  including  the  Philippine  Islands,  Africa,  and  is  also  met  with 
in  Europe,  in  the  southern  parts  of  the  Balkan  peninsula,  and  in 
southern  Russia.  The  organism  causing  the  disease  is  not  known, 
but  it  is  probably  of  a  type  similar  to  that  of  contagious  pleuro- 
pneumonia.  It  appears  that  the  virus  contained  in  natural  albumin- 
ous body  fluids  does  not  pass  the  Chamberland  or  Berkefeld  filters, 
but  that  it  does  when  such  albuminous  fluids  are  diluted  with  a  sterile 
watery  fluid. 

Pathologic  Lesions. — The  most  characteristic  pathologic  lesions  of 
the  disease  which  has  a  rapid  onset  are  strong  hyperemia  of  the  con- 
junctiva, fetid  discharge  from  the  nostrils,  and  hemorrhagic  diar- 
rhea. On  postmortem  examination  the  fourth  stomach  is  found 
strongly  hyperemic,  with  petechise  on  the  elevated  margins  of  the 
folds  of  the  mucosa,  and  in  the  more  chronic  cases  ulcerations  are 
formed.  The  intestines  are  covered  with  a  fibrinous  exudate,  which 


444  CATTLE  PLAGUE 

sometimes  forms  a  complete  cast  of  the  gut.  Peyer's  patches  and 
the  solitary  follicles  are  swollen  and  hyperemic.  Sometimes  the  serous 
membranes,  particularly  the  pericardium  and  the  pleura,  likewise 
show  hemorrhages.  The  liver  and  spleen  are  not  markedly  enlarged, 
though  much  congested.  In  certain  localities  epizootics  have  occasion- 
ally also  shown  hemorrhages  and  ulcerations  in  the  buccal  and  nasal 
mucosa. 

Immunization. — Since  it  had  been  noticed  that  animals  which  had 
recovered  from  the  disease  were  immune,  attempts  at  immunization 
were  early  undertaken.  The  first  experiments  of  this  kind  were  made 
by  Semmer  and  Nencki.  Koch,  Kolle  and  Turner,  and  Theiler  and 
others  have  worked  out  methods  of  preparing  a  "Rinderpest"  immune 
serum  of  high  value.  This  is  done  in  the  following  manner :  Healthy 
cattle  which  must  particularly  be  free  from  piroplasmosis  and  try- 
panosomiasis  are  first  immunized  by  the  simultaneous  method.  On  one 
side  of  the  body  they  receive  1  c.c.  of  virulent  blood  from  an  animal 
sick  with  cattle  plague  and  on  the  other  side  a  dose  of  15  to  55  c.c.  of  an 
immune  serum.  An  immune  serum  of  high  value  is  subsequently  fur- 
nished only  by  those  animals  which  react  to  this  simultaneous  injection 
by  fever  and  otherwise.  Races  of  cattle  which  show  no  reaction  are 
unsuitable  for  the  preparation  of  an  immune  serum.  After  an 
animal  which  has  reacted  has  recovered  it  receives  an  injection  of 
100  c.c.  of  virulent  blood.  This  is  again  followed  by  fever.  When 
the  animal  has  fully  recovered  it  receives  200  c.c.  of  virulent  blood. 
This  method  of  giving  gradually  increasing  doses  is  continued  until 
1000  c.c.  of  virulent  blood  have  been  injected  at  one  time.  After  the 
last  reaction  has  subsided  the  animal  is  bled  on  three  successive  days 
or  on  three  days  with  an  interval  of  one  day  between.  Each  time 
4500  c.c.  are  withdrawn.  After  a  short  interval  the  animal  can 
again  be  injected  with  a  large  dose  of  virulent  blood  and  also  be  bled 
again.  In -this  manner  it  is  possible  to  obtain  large  amounts  of  the 
immune  blood.  The  latter  is  allowed  to  coagulate,  the  serum  is 
collected  and  a  sufficient  quantity  of  5  per  cent,  carbolic  acid  is  added 
to  it  so  that  the  mixture  contains  0.5  per  cent,  of  carbolic  acid.  In 
the  preparation  of  the  immune  serum  it  is  necessary  always  to  have 
on  hand  fresh  virulent  blood.  This  is  obtained  by  injecting  0.1  to 
0.05  c.c.  of  very  virulent  blood  into  unprotected  but  perfectly  healthy 
cattle.  Koch  has  shown  that  sheep  are  also  susceptible  to  cattle 
plague  if  inoculated  with  virulent  blood,  though  they  rarely  die  from 
the  disease,  and  for  this  reason  they  may  be  used  to  furnish  the 
virulent  blood.  The  titre,  or  immunizing  value  of  the  immune  serum, 
is  determined  by  injecting  non-immunes  with  1  c.c.  of  virulent  blood 
and  estimating  what  quantity  of  the  immune  serum  will  protect 
against  this  dose. 

Kolle  and  Turner  have  ascertained  that  a  good  immune  serum  will 
protect  cattle  for  three  months  against  "Rinderpest"  in  a  dose  of  100 
to  200  c.c.,  and  that,  particularly  in  still  larger  doses,  it  will  save  cattle 


FOOT-AND-MOUTH  DISEASE  445 

already  in  the  early  stage  of  the  disease.  Immune  serum,  if  properly 
preserved,  retains  its  protective  and  curative  properties  for  several 
years ;  in  one  observation  for  five  years. 


AFRICAN  HORSE  SICKNESS. 

This  affection,  also  known  as  pestis  equorum,  "Pferdepest" 
(German),  peste  du  cheval  (French),  is  an  acute  or  subacute  disease 
of  horses,  prevalent  in  epizootic  form  in  Africa.  As  first  demonstrated 
by  McFadyean  and  afterward  by  Nocard,  it  is  due  to  an  ultra- 
microscopic,  filterable  virus.  Theiler,  Eddington,  and  Koch  have 
studied  the  disease,  with  the  object  of  preparing  a  protective  serum. 
The  most  important  pathologic  changes  of  the  disease  are  gelatinous 
infiltrations  of  the  subcutaneous  and  intermuscular  connective  tissue, 
swelling  of  the  superficial  lymph  glands,  very  marked  catarrhal 
swelling  of  the  mucosa  of  the  stomach,  and  the  first  portion  of  the 
small  intestine,  edema  of  the  lungs,  and  occasionally  the  formation 
of  gelatinous  deposits  on  the  pleura,  pericardium,  and  epicardium. 
One  attack  of  the  disease  produces  a  relative  immunity  only,  and 
animals  which  have  recovered  from  the  disease  may  again  be  made 
very  sick  by  the  injection  of  large  doses  of  virulent  blood.  Koch  used 
horses  which  had  passed  through  a  natural  infection  (so-called  "salted" 
horses)  in  the  preparation  of  an  immune  serum  of  high  value.  It  is 
obtained  by  injecting  into  "salted"  horses  increasing  doses  of  virulent 
blood  obtained  from  sick  horses  shortly  before  death.  According  to 
Theiler  the  protective  inoculation  consists  in  the  injection  of  300  c.c. 
immune  serum  into  the  jugular  vein  and  of  a  small  amount  of  virulent 
blood  under  the  skin. 


FOOT-AND-MOUTH  DISEASE. 

Foot-and-mouth  disease,  also  known  as  aphthae  epizooticse, 
aphthous  fever,  infectious  aphtha,  eczema  epizootica,  "Maul  und 
Klauenseuche"  (German),  "fievre  aphtheuse"  (French),  is  an  acute 
highly  contagious  fever,  due  to  a  specific  contagium.  It  is  character- 
ized by  the  eruption  of  vesicles  or  blisters  in  the  buccal  mucosa, 
around  the  coronets  of  the  feet,  and  between  the  toes.  It  is  most 
commonly  a  disease  of  cattle,  but  it  also  affects  swine,  sheep,  goats, 
buffalo,  bison,  deer,  etc.,  and  less  frequently  horses,  dogs,  and  cats. 
Man  may  be  infected  by  contact  with  sick  animals  or  by  drinking 
their  raw  milk. 

The  contagium,  an  ultramicroscopic  filterable  virus,  is  contained  in 
the  contents  of  the  vesicles,  in  the  saliva  and  milk,  and  during  the 
height  of  the  disease,  also  in  the  blood  serum.  While  the  mortality 
from  the  disease  is  generally  not  very  high,  epidemics  are  so  wide- 


446  FOOT-AND-MOUTH  DISEASE 

spread  and  lead  to  so  considerable  a  loss  in  milk  that  the  total  loss 
is  relatively  large.  The  disease  is  very  prevalent  in  Europe  and  has, 
on  several  occasions,  invaded  the  United  States.  It  may  enter  a  free 
territory  in  a  very  insidious  manner  and  spread  before  it  has  been 
recognized.  The  most  recent  outbreak  in  this  country,  discovered  in 
October,  1908,  by  Pearson,  in  Pennsylvania,  and  described  in  Circular 
147  of  the  Bureau  of  Animal  Industry,  by  Mohler  and  Rosenau, 
furnishes  an  interesting  instance  of  the  mode  of  spreading.  The 
disease  was  found  to  have  been  introduced  into  the  United  States 
with  a  smallpox  vaccine  imported  from  Japan.  The  vaccine  had 
been  used  and  propagated  by  one  manufacturing  firm  for  a  con- 
siderable time  and  no  outbreak  of  hoof-and-mouth  disease  occurred, 
because  the  inoculated  calves  were  slaughtered  immediately  after 
the  vaccine  had  been  obtained.  A  second  firm,  however,  afterward 
obtained  vaccine  from  the  first,  used  it  in  the  inoculation  of  calves, 
and  sold  these  animals  after  the  crop  of  lymph  had  been  collected. 
It  was  through  these  calves  that  the  hoof-and-mouth  disease  had 
been  spread  to  a  number  of  places  before  it  was  discovered.  For- 
tunately the  Government  authorities  traced  all  the  foci  of  infection  and 
stamped  out  the  epizootic  before  it  had  reached  any  great  proportions. 
The  conclusions  drawn  by  Mohler  and  Rosenau  and  the  others  who 
took  part  in  the  investigation  are  the  following: 

1.  The  recent  outbreak  of  foot-and-mouth  disease  in  this  country 
started  from  some  calves  used  to  propagate  vaccine  virus. 

2.  The  vaccine  virus  used  on  these  calves  has  been  proved  to 
contain  the  infection  of  foot-and-mouth  disease. 

3.  The  outbreaks  of  foot-and-mouth  disease  in  1902-3  probably 
had  a  similar  origin. 

4.  It  is  probable  that  the  foot-and-mouth  infection  got  into  the 
vaccine  virus  in  some  foreign  country  in  which  the  disease  prevailed, 
and  was  introduced  into  the  United  States  through  the  importation 
of  this  contaminated  vaccine. 

5.  The  symbiosis  between  the  infections  of  vaccinia  and  foot-and- 
mouth  disease  is  especially  interesting.    Animals  vaccinated  with  the 
mixed  virus,  as  a  rule,  show  only  the  lesions  of  one  of  these  diseases, 
namely,  vaccinia;  nevertheless  the  infectious  principle  of  foot-and- 
mouth  disease  remains  in  the  vaccinal  eruption. 

Protective  Inoculation. — This  has  been  practised  empirically  for  a 
long  time.  Since  the  disease  spreads  through  a  large  herd  rather 
slowly,  and  may  thus  cause  a  prolonged  quarantine,  stock  owners 
have  been  in  the  habit  of  inoculating  their  cattle  after  an  outbreak 
by  rubbing  saliva  from  infected  animals  into  the  mucous  membrane  of 
healthy  ones,  or  contaminating  rough  fodder  with  the  infected  saliva. 
This  method,  however,  is  only  applicable  when  the  epizootic  occurs 
in  a  milder  form.  The  severe  form  of  the  disease,  when  propagated 
in  this  manner,  is  likely  to  cause  considerable  loss.  Loeffler,  Frosch, 
Uhlenhuth  and  Hecker,  and  others,  have,  for  a  number  of  years,  been 


FOWL  PLAGUE  447 

engaged  in  attempts  to  devise  an  effective  method  of  protecting 
cattle  against  hoof-and-mouth  disease  by  injecting  simultaneously  an 
immune  serum  and  virulent  lymph,  but  up  to  the  present  time  no 
efficient  method  seems  to  have  been  worked  out.  The  last  method 
recommended  by  Loeffler  requires  four  different  inoculations.  Casper, 
who  has  reviewed  the  subject,  concludes  that  there  is  yet  no  method 
of  appreciable  value  in  practice,  and  that  the  whole  subject  is  still  in 
an  experimental  stage. 


DISTEMPER  IN  DOGS. 

Distemper  in  dogs,  dog  plague,  dog  ill,  "Hundeseuche"  (German), 
Pasteurellosis  canum,  "Maladie  du  jeune  age"  (French),  is  an  acute, 
contagious  disease  of  young  carnivora,  particularly  of  dogs,  character- 
ized by  an  acute  catarrh  of  the  upper  respiratory  passages,  often 
associated  with  a  catarrhal  pneumonia.  In  fatal  cases  the  mucous 
membranes  of  the  nose,  larynx  and  bronchi  are  reddened,  swollen 
and  covered  with  an  abundant  fibrinous  exudate.  The  finest  bron- 
chioles are  filled  with  a  purulent  material  and  the  pulmonary  paren- 
chyma exhibits  smaller  or  larger  areas  of  consolidation.  The  pleura 
of  such  diseased  portions  of  lung  is  often  covered  with  a  slight  fibrin- 
ous exudate.  The  gastric  and  intestinal  mucosa  is  swollen  and 
reddened,  the  mediastinal  and  mesenteric  glands  are  swollen.  The 
heart,  liver,  and  kidneys  show  evidences  of  parenchymatous  degen- 
eration. In  some  cases  the  spinal  cord  shows  disseminated  inflam- 
matory foci  (myelitis  disseminata).  Ligniere  claimed  to  have  dis- 
covered a  bipolar  bacillus  (Pasteurella  canis)  as  the  cause  of  dog 
distemper,  but  his  claim  has  not  been  confirmed.  Jess  also  claimed 
the  discovery  of  a  bacillus  as  the  cause  of  distemper,  but  his  work 
likewise  has  not  been  confirmed.  It  appears  rather  that  the  disease  is 
due  to  an  ultramicroscopic  filterable  virus.  None  of  the  vaccines  and 
sera  against  dog  distemper  which  have  from  time  to  time  appeared 
have  proved  efficient. 

FOWL  PLAGUE. 

Fowl  plague,  pestis  gall  ina  rum,  pestis  avium,  "Huhnerpest" 
(German),  "peste  aviaire"  (French),  is  an  acute  contagious  disease 
of  chickens,  which  has  formerly  generally  been  confounded  with 
chicken  or  fowl  cholera.  It  is,  however,  a  distinct  disease,  not  due, 
like  chicken  cholera,  to  a  bacterium  of  the  hemorrhagic  septicemia 
group,  but  to  an  ultramicroscopic  filterable  virus.  The  latter  is 
present  in  the  blood,  the  secretion  from  the  nose,  the  feces,  the 
serous  exudates,  and  the  bile  of  sick  birds.  Infection  can  be  pro- 
duced artificially  by  inoculation  of  exceedingly  small  doses  of  blood 
(0.000001  c.c.).  Blood  kept  in  fused  glass  tubes  in  a  cool,  dark 


448  CHICKEN-POX 

place  can  retain  its  virulency  for  three  months.  In  very  acute  cases 
of  this  disease  which  has  been  observed  in  several  European  countries 
the  only  pathologic  changes  found  are  hemorrhages  into  the  serous 
membranes;  in  less  acute  cases  there  is  a  marked  edema  of  the 
subcutaneous  connective  tissues  and  a  collection  of  pale  yellow  fluid 
into  the  serous  membranes  and  a  fibrinous  exudate  upon  them. 
The  disease,  therefore,  has  also  been  called  typhus  exudativus 
gallinarum. 

CHICKEN-POX. 

Chicken-pox,  epithelioma  contagiosum  avium,  "  Gefliigelpocke " 
(German),  "la  petite  verole"  (French),  is  a  chronic  contagious  disease 
of  fowl,  characterized  by  the  formation  of  epithelial  nodules  on  the 
comb  and  neck  and  on  the  mucous  membranes  of  the  head.  It  is 
caused  by  a  filterable  virus.  The  latter  can  be  obtained  by  grinding 
up  the  epithelial  proliferations,  mixing  the  mass  with  some  sterile 
physiologic  salt  solution,  and  filtering  the  mixture  through  porcelain 
or  clay  filters.  The  disease,  after  natural  infection,  begins  with  the 
formation  of  grayish-red,  shiny  patches  in  the  places  indicated. 
The  small,  slightly  elevated  patches  soon  increase  in  size,  become 
more  elevated  and  grayish  yellow  in  color.  They  finally  form  nodules 
of  the  size  of  a  pea  or  larger,  dry  and  warty  in  character,  and  con- 
taining in  their  interior  a  yellowish  fatty  mass.  The  wart-like  masses 
sometimes  become  confluent  and  cover  entirely  larger  areas  of  skin 
or  mucosa.  The  disease  generally  ends  in  recovery  by  drying  up 
and  shedding  of  the  epithelial  proliferations.  If,  on  the  other  hand, 
the  affected  animals  die  it  is  found  that  the  process  has  extended 
from  the  mouth  toward  the  larynx  and  in  it  and  the  bronchi  a  muco- 
purulent  exudate  and  caseous  lumps  are  found.  The  lungs  contain 
bronchopneumonic  inflammatory  foci.  The  intestinal  mucosa  is 
inflamed  and  studded  with  small  hemorrhages.  The  histologic 
changes  of  the  skin  and  mucous  membranes  of  the  mouth  consist 
in  a  hypertrophy  of  the  epithelial  covering,  a  proliferation  of  the 
interpapillary  pegs  with  congestion  of  vessels  and  inflammatory  cell 
infiltration  in  the  derma. 

Bollinger  and  Coskor  claim  that  epithelioma  contagiosum  of  man 
and  of  fowl  is  identical  and  that  it  can  be  experimentally  transferred 
from  the  former  to  the  latter.  The  disease  described  and  cotnmonly 
known  as  chicken-pox  in  fowls  has,  of  course,  nothing  in  common 
with  the  disease  known  as  chicken-pox  in  man,  the  latter  being  one 
of  the  febrile  exanthematous  contagious  diseases,  characterized  by  a 
vesicular  eruption  over  the  entire  body. 

' 


QUESTIONS  449 


QUESTIONS. 

1.  What  is  contagious  pleuropneumonia  in  cattle?    Where  does  the  disease 
occur?    Has  it  ever  been  seen  in  the  United  States? 

2.  Describe  its  pathologic  lesions  in  the  lungs. 

3.  Describe  its  changes  produced  in  the  pleura. 

4.  What  is  meant  by  a  carneous  lung? 

5.  Which  material  contains  the  infectious  virus? 

6.  Describe  the  formation  of  necrotic  pulmonary  sequesters  in  the  disease. 

7.  Describe  the  histo-pathologic  changes  in  the  lungs  and  pleura. 

8.  What  is  a  pleuritis  sicca? 

9.  Describe  the  method  of  obtaining  clear,  uncontaminated  virulent  serous 
fluid. 

10.  What  does  microscopic  examination  of  this  fluid  show? 

11.  How  can  the  organism  of  pleuropneumonia  be  cultivated? 

12.  How  do  its  cultures  look  in  collodion  sacs?    How  on  Martin's  gelatin-serum 
medium? 

13.  How  is  the  latter  prepared? 

-14.  Under  what  conditions  does  the  virus  of  pleuropneumonia  pass  a  Cham- 
berland  filter?  under  what  conditions  does  it  not  pass? 

15.  Discuss  protective  inoculation  against  pleuropneumonia. 

16.  What  is  Rinderpest? 

17.  What  kind  of  an  organism  causes  it? 

18.  Describe  the  most  characteristic  pathologic  lesions  of  the  disease. 

19.  Describe  the  Kolle-Turner  method  of  preparing  a  rinderpest  immune 
serum  of  high  value. 

20.  What  method  is  used  to  have  on  hand  large  amounts  of  virulent  blood? 

21.  What  are  the  curative  properties  of  a  high- value  immune  serum?   What  are 
its  properties  as  to  stability? 

22.  What  is  African  horse-sickness  due  to?    What  are  its  most  characteristic 
pathologic  lesions? 

23.  What  is  hoof-and-mouth  disease  in  cattle? 

24.  How  may  the  disease  be  spread  by  cowpox  vaccine  lymph  ?  • 

25.  What  methods  of  protective  inoculation  have  been  practised?     Discuss 
their  value. 

26.  What  is  the  cause  of  distemper  in  dogs?    What  are  the  most  characteristic 
anatomic  lesions? 

27.  What  is  the  cause,  what  are  the  pathologic  lesions  of  fowl  plague? 

28.  Describe  the  pathologic  lesions  of  chicken-pox,  or  epithelioma  contagiosum 
avium? 


29 


PART   III. 
MICROORGANISMS  IN  FOODS  AND  SOILS, 


CHAPTER    XLIL 

THE  BACTERIA  OF  MEAT-POISONING—BACILLUS  ENTERITIDIS— 
BACILLUS  BOTULINUS. 

MEAT  from  healthy  animals,  obtained  in  a  clean  manner,  is  free 
from  bacteria.  At  first  they  are  found  on  the  external  surfaces  only, 
and  after  a  variable  period  of  time,  depending  upon  temperature 
and  other  environmental  conditions,  they  may  penetrate  to  a  certain 
distance  into  the  interior  of  the  meat.  If  kept  for  a  longer  period  and 
at  a  higher  temperature,  putrefactive  saprophytic  bacteria  develop 
throughout  and  cause  it  to  become  putrid.  Such  meat  may  often  be 
ingested  by  animals  and  occasionally  by  man  without  necessarily 
producing  disease.  On  the  other  hand,  meat  from  animals  which 
have  been  slaughtered  on  account  of  disease  may  appear  in  every  way 
normal,  and  yet  may  produce  violent  disease  and  even  death  when 
eaten.  In  some  cases  only  raw  or  very  insufficiently  cooked  or  boiled 
meat  produces  disease,  but  in  other  cases  disease  also  follows  the 
ingestion  of  meat  which  has  been  well  cooked.  The  study  of  meat- 
poisoning  epidemics  in  various  parts  of  the  world  has  shown  that 
sickness  generally  follows  the  ingestion  of  meat  from  animals  suffering 
from  severe  gastro-intestinal  disturbances  or  general  septic  conditions. 
In  a  number  of  examinations  made  on  meat  of  this  kind  the  blood- 
vessels were  found  full  of  bacteria,  which  when  obtained  in  pure 
cultures  proved  highly  pathogenic  to  animals.  Bellinger  was  prob- 
ably the  first  observer  who  held  that  such  intoxication  after  the  use 
of  meat  was  due  to  the  intestinal  septic  conditions  from  which  the 
animal  that  furnished  the  meat  had  suffered  itself. 


BACILLUS  ENTERITIDIS  OF  GARTNER. 

Morphology  and  Historical. — Gartner  was  the  first  to  isolate  a 
bacterium  as  the  cause  of  a  meat-poisoning  epidemic  following  the 
use  of  meat  from  an  emergency  slaughtered  sick  cow.  Since  then 


452  THE  BACTERIA  OF  MEAT-POISONING 

Van  Ermengem,  Gaffky  and  Paak,  Neelsen  and  Johne,  and  others, 
have  also  studied  similar  outbreaks.  They  found  a  bacillus  acting 
through  a  poison  which  is  not  even  destroyed  by  boiling  the  infected 
meat,  as  the  cause  of  a  certain  form  of  meat-poisoning.  This  organism 
is  known  as  the  Bacillus  enteritidis  of  Gartner.  It  belongs  to  the 
typhoid-colon  group.  It  is  a  short  bacterium,  and  is  frequently  of 
ovoid  form,  not  longer  than  0.2  to  0.4  micron,  and  generally  arranged 
in  pairs.  It  often  stains  unequally,  more  at  the  poles,  and  then 
resembles'  an  organism  of  the  hemorrhagic  septicemia  group.  It  is 
Gram  negative,  motile,  and  provided  with  four  to  eight,  sometimes 
ten  or  twelve  long  flagella.  These  cannot  be  well  stained  by  Loeffler's 
method,  but  they  can  be  demonstrated  by  the  silvering  method  of 
Van  Ermengem. 

Cultural  Properties. — The  superficial  colonies  on  gelatin  are  variable 
in  form  and  often  cannot  be  distinguished  from  the  colonies  of  the 
Bacillus  coli,  though  they  are  generally  more  transparent  and  more 
regular  at  the  periphery.  The  organism  does  not  form  indol  or  in 
traces  only.  It  does  not  coagulate  milk,  but  causes  it  to  become 
decidedly  alkaline  after  ten  days,  and  also  more  transparent  and 
yellowish  in  color.  It  ferments  glucose,  lactose,  galactose,  maltose, 
saccharose,  and  also  glycerin.  Bouillon  becomes  rapidly  clouded,  and 
an  easily  torn  pellicle  is  formed  on  the  surface.  It  does  not  produce 
a  fecal  smell  like  the  colon  bacillus.  The  growth  on  potatoes  varies. 
It  is  sometimes  invisible  like  that  of  the  typhoid  bacillus,  at  other 
times  dirty  yellowish  or  brownish  like  that  of  the  colon  bacillus.  It 
grows  quite  well  in  Petruschky's  litmus  whey  without  much  acid 
production  or  color  changes.  In  Rotberger's  neutral-red  agar, 
containing  0.3  per  cent,  glucose,  the  color  disappears  and  gas  is 
produced  after  eighteen  to  twenty-four  hours.  On  the  Drigalski- 
Conradi  medium  bluish  colonies  somewhat  larger  and  less  transparent 
than  those  of  the  tyhoid  bacillus  are  formed  after  sixteen  to  eighteen 
hours. 

Toxin  Production  and  Other  Properties. — The  Bacillus  enteritidis  is 
distinguished  from  all  other  bacteria  of  the  typhoid-colon  group  by 
its  property  of  forming  diffusible  toxins,  which  are  very  resistant  to 
higher  temperatures.  The  bacilli,  when  introduced  in  small  quan- 
tities into  the  stomachs  of  small  animals  or  injected  subcutaneously 
intravenously  or  intraperitoneally  have  a  fatal  effect.  Such  susceptible 
animals  are  mice,  guinea-pigs,  rabbits,  monkeys,  calves,  etc.  The 
organism  produces  local  inflammatory  conditions  in  the  serous 
membranes,  and  also  a  general  septicemia,  and  it  can  be  found  in 
considerable  numbers,  particularly  in  the  muscles.  The  toxin,  even 
if  it  has  been  heated  to  the  boiling  point  of  water,  produces  the  same 
pathologic  lesions  in  the  intestines,  the  kidneys,  and  in  the  serous 
membrane  in  general.  Whether  the  Bacillus  enteritidis  is  a  single 
species  or  whether  there  are  several  nearly  related  varieties  is  a  point 
not  yet  fully  settled.  It  appears  that  at  least  one  of  the  varieties  of 


PROPHYLACTIC  MEASURES  453 

meat-poisoning  bacilli  described  belongs  to  the  group  of  paratyphoid 
bacilli.  These  are  several  varieties  of  bacilli  which  closely  resemble 
the  Bacillus  typhosus. 

Agglutination. — It  has  been  shown  that  the  blood  serum  of  patients 
suffering  from  meat  poisoning  has  a  comparatively  high  agglutinating 
power  of  1  to  75-400  toward  the  Bacillus  enteritidis,  isolated  from  the 
meat  which  had  caused  the  sickness,  while  the  serum  from  healthy 
persons  agglutinates  in  a  proportion  of  1  to  25  only.  It  has,  on  the 
other  hand,  also  been  demonstrated  that  the  serum  from  typhoid 
hyperimmunized  animals  also  agglutinated  the  Bacillus  enteritidis  in 
very  high  dilutions,  but  it  is  never  agglutinated  in  as  high  dilutions  as 
the  typhoid  bacillus  itself.  De  Noble  observed  that  a  typhoid  immune 
serum  which  agglutinated  the  typhoid  bacillus  in  1  to  30,000  would 
agglutinate  the  Bacillus  enteritidis  in  1  to  2000.  Fischer  showed 
that  an  immune  serum  of  the  Bacillus  enteritidis  which  would  agglu- 
tinate it  in  1  to  40,000  would  agglutinate  the  Bacillus  coli  only  in 
1  to  10.  This  shows  that  the  Bacillus  enteritidis  is  certainly  not  a 
variety  of  the  colon  bacillus.  Various  stems  of  hog-cholera  bacilli 
behave  toward  such  an  immune  serum  like  very  distinct  varieties  and 
not  like  one  identical  species. 

Infection. — Infection  with  the  Bacillus  enteritidis  may  occur 
through  other  sources  than  meat  from  sick  animals  containing  this 
organism.  Probably  it  may  also  be  conveyed  by  milk  and  by  contact 
between  sick  and  healthy  persons.  It  appears  also  that  an  organism 
described  by  Petruschky  under  the  name  of  Bacillus  fecalis  alkaligenes 
obtained  from  the  feces  of  a  patient  sick  with  a  typhoid-like  disease 
is  identical  with  the  Bacillus  enteritidis. 

Prophylactic  Measures. — To  protect  the  public  against  the  use  of 
meat  which  might  cause  infection  with  the  Bacillus  enteritidis  the 
following  measures  are  recommended:  Small  pieces  of  meat  are 
removed  with  sterile  instruments  2  or  3  cm.  below  the  external  surface. 
With  these  pieces  agar  tubes  are  inoculated.  They  should  remain 
sterile,  since  the  interior  of  meat  from  healthy  animals  does  not  contain 
any  bacteria.  Another  method  was  proposed  by  De  Noble.  A  piece 
of  meat  is  obtained  from  the  interior  and  its  juice  is  expressed  and 
diluted  to  the  proportion  of  1  to  10  to  20  with  physiologic  salt  solution. 
With  this  fluid  agglutination  tests  are  made  with  emulsions  of  cultures 
of  the  Bacillus  enteritidis.  If  the  juice  agglutinates  in  a  dilution  of 
1  to  10  to  20  it  shows  enteritidis  infection  of  the  meat,  because  the 
juice  from  healthy  meat  does  not  agglutinate  in  a  proportion  of  1  to  1. 
If  cultures  are  to  be  obtained  from  infected  meat  it  is  well  to  keep  it 
for  twenty-four  hours  at  18°  to  20°  C.  Under  these  conditions  the 
enteritidis  bacillus  multiplies  rapidly  in  the  interior  of  the  infected 
meat. 

Meat  poisoning  has  also  been  observed  from  the  use  of  meat  already 
somewhat  putrid  and  containing  large  numbers  of  colon  or  proteus 
bacilli. 


454  BACILLUS  BOTULINUS 


BACILLUS  BOTULINUS. 

There  is  still  another  form  of  meat  poisoning  due  to  a  bacterium, 
the  toxins  of  which  are  destroyed  by  heating.  This  form  of  meat 
poisoning  is  known  as  botulismus,  or  allantiasis.  It  has  been  most 
frequently  observed  following  the  ingestion  of  sausages  and  pickled 
or  smoked  meat.  Similar  affections  have  been  traced  to  the  con- 
sumption of  fish  and  they  are  then  spoken  of  as  ichthyosismus.  Articles 
of  food  through  which  these  intoxications  are  produced,  are  generally 
prepared  and  kept  under  conditions  which  favor  the  development  of 
anaerobic  bacteria;  they  are  consumed  raw  or  imperfectly  boiled  or 
cooked.  The  symptoms  of  botulism  differ  from  the  gastro-intestinal 
disturbances  characteristic  of  Bacillus  enteritidis  infection  or  intoxica- 
tion and  point  to  nervous  disturbances,  such  as  paralysis  of  the  muscles 
of  the  eye,  double  vision,  constipation,  and  enuresis.  Diarrhea  and 
vomiting  are  rare,  and  if  present  they  are  of  a  transitory  character. 
Other  disturbances  often  noticed  are  dryness  of  the  buccal  and 
pharyngeal  mucosa  and  redness,  disturbance  of  the  voice,  and 
difficulty  in  respiration. 

Morphology. — The  Bacillus  botulinus  was  discovered  in  1895  in  a 
meat-poisoning  epidemic  affecting  50  persons  by  Van  Ermengem,  who 
investigated  it  most  thoroughly  and  furnished  the  following  descrip- 
tion: It  is  a  strictly  anaerobic  bacillus,  4  to  6  micra  long  and  0.9  to 
1.2  wide;  its  ends  are  somewhat  rounded  off,  it  forms  groups  of  two 
or  somewhat  longer  chains;  it  is  sluggishly  motile  and  possesses  four 
to  eight  very  fine  flagella,  arranged  in  a  peritrichous  manner. 

Cultural  and  Staining  Properties. — It  stains  easily  and  is  Gram 
positive.  The  growth  is  most  characteristic  on  glucose  gelatin  plates, 
where  the  young  colonies  are  circular,  transparent,  slightly  yellowish, 
and  composed  of  coarse  granules,  which  show  a  constant  motility. 
Zones  of  liquefied  gelatin  surround  the  colonies.  The  colonies  later 
become  larger,  brown  in  color,  and  opaque,  and  only  the  granules  at 
the  periphery  retain  their  motility  and  look  like  thorny  projections. 
The  latter  become  larger  and  divide  and  form  digit-like  masses  and 
the  entire  colony  resembles  a  sunflower.  Stick  or  stab  cultures  in 
glucose  gelatin  or  agar  are  not  as  characteristic.  These  media  become 
cleft  and  broken  up  in  consequence  of  abundant  gas  formation.  The 
liquefied  gelatin  later  becomes  transparent  because  the  growth  falls  to 
the  bottom  as  a  whitish,  flocculent  sediment.  A  large  amount  of  gas 
composed  of  hydrogen  and  methane  (CH4)  is  formed.  All  cultures 
give  off  a  smell  of  butyric  acid,  but  there  is  no  marked  fetor.  Milk 
is  not  coagulated;  lactose  and  saccharose  are  not  fermented.  The 
growth  is  abundant  under  anaerobic  conditions  between  18°  to  25°  C. ; 
at  37°  to  38.5°  C.  the  growth  is  scanty,  involution  forms  soon  appear, 
and  toxins  are  no  longer  formed.  In  bouillon  cultures  kept  at  37°  to 
38.5°  C.  the  organism  forms  long,  intertwined  filaments.  The  bacillus 
at  medium  temperatures  forms  oval,  somewhat  elongated,  endogenous 


TOXIN  PRODUCTION  455 

spores.  It  requires  an  alkaline  medium,  no  growths  occur  on  acid 
media,  and  5  to  6  per  cent,  of  sodium  chloride  also  prevents  its  multipli- 
cation. The  spores  are  not  very  resistant  and  are  destroyed  if  exposed 
one  hour  to  80°  C.  They  are  killed  by  5  per  cent,  carbolic  acid  within 
twenty-four  hours.  If  protected  against  air  and  sunlight  they  remain 
alive  for  a  long  time  either  in  moist  or  desiccated  condition.  The 
organism,  however,  dies  comparatively  quickly  in  glucose  gelatin  and 
the  cultures  must  be  transferred  every  three  or  four  weeks.  It  is 
also  necessary  to  transplant  a  larger  mass  of  the  sediment  from  such 
cultures.  The  culture  media  should  always  be  distinctly  alkaline 
and  not  exposed  to  temperatures  above  25°  C. 

Animals  Susceptible.— If  small  amounts  of  cultures  (0.0003  to  0.001) 
are  injected  into  rabbits  the  animals  may  die  within  thirty-six  to 
forty-eight  hours.  Sometimes  they  live  three  to  four  days,  then 
become  paralytic  and  die  shortly  after  these  symptoms  have  appeared. 
If  the  cultures  are  introduced  into  the  stomach  the  effect  is  slow  and 
uncertain.  Doses  of  5  to  10  c.c.  of  a  bouillon  culture  may  kill  rabbits 
within  forty-eight  hours.  At  times  a  slow  cachexia  develops,  and  the 
animals  only  die  after  a  number  of  weeks.  Guinea-pigs  are  very  sus- 
ceptible to  minimal  doses ;  they  show  aphonia,  forced  respiration,  great 
dyspnea,  and  death.  Mice  and  monkeys  are  not  very  susceptible. 
Dogs,  chickens,  and  cats  are  refractory,  and  can  withstand  large  doses 
given  repeatedly,  but  they  may  show  some  transitory  paretic  symptoms. 

Toxin  Production. — Van  Ermengem  believes  that  the  symptoms  due 
to  the  Bacillus  botulinus  in  man  and  in  artificial  animal  inoculation 
are  due  exclusively  to  intoxication  without  any  multiplication  of  the 
bacillus  in  the  infected  organism.  He  classifies  the  Bacillus  botulinus 
as  a  pathogenic  or  toxigenic  saprophyte,  and  likens  its  action  to  that 
of  the  large  poisonous  fungi  which  produce  disease  and  death  by  the 
amount  of  already  formed  toxin  introduced  with  them  into  a  suscept- 
ible animal.  The  botulismus  toxin  which  affects  susceptible  animals 
not  only  in  subcutaneous  or  intravenous  injections,  but  also  if  intro- 
duced into  the  stomach,  is  not  very  resistant.  It  is  destroyed  if 
heated  to  80°  C.  for  one  hour;  it  is  almost  instantaneously  destroyed 
by  a  3  per  cent,  carbonate  of  soda  solution;  it  is  somewhat  more 
resistant  toward  acid  and  soon  succumbs  to  the  effect  of  light  and 
air.  This  toxin,  like  the  tetanus  toxin,  is  fixed  by  emulsions  from 
the  central  nervous  system,  by  lecithin,  cholesterin,  fatty  substances. 
Van  Ermengem  recommends  the  following  as  prophylactic  measures 
against  infection  with  the  Bacillus  botulinus:  Articles  of  food  which 
in  a  raw  condition  may  be  easily  decomposed,  such  as  ham,  sausages, 
salted  fish,  should  not  be  eaten  raw.  All  foods  which  give  off  a  smell 
of  butyric  acid  are  suspicious  and  may  contain  the  Bacillus  botulinus. 
Pickled  meat  or  fish  should  be  soaked  before  use  in  a  not  less  than 
10  per  cent,  salt  solution,  which  will  destroy  the  Bacillus  botulinus. 

Kempner  has  prepared  an  antitoxic  botulinus  serum  which  proved 
efficacious  in  protecting  animals. 


456  BACILLUS  BOTULINUS 


QUESTIONS. 

1.  Discuss  the  bacterial  contents  in  meat  from  healthy  animals. 

2.  Under  what  circumstances  are  bacteria  found  in  the  vessels  in  the  interior 
of  the  meat  of  food  animals  ? 

3.  May  such  bacteria  containing  meat  be  dangerous  after  cooking? 

4.  Who  discovered  the  first  meat  poison  organism? 

5.  Describe  its  morphology. 

6.  Describe  its  cultural  characteristics. 

7.  Differentiate  between  the  cultural  features  of  Bacillus  enteritidis  and 
Bacillus  coli. 

8.  What  symptoms  does  the  Bacillus  enteritidis  cause  in  man  if  ingested 
with  infected  meat? 

9.  What  is  the  behavior  of  the  blood  serum  of  persons  suffering  from  Bacillus 
enteritidis  infection  toward  this  organism? 

10.  How  does  a  typhoid-immune  serum  of  very  high  value  behave  toward 
the  Bacillus  typhosus  and  the  Bacillus  enteritidis? 

11.  What  is  the  Bacillus  fecalis  alkaligenes? 

12.  What  methods  have  been  recommended  to  detect  the  infection  of  meat 
with  the  Bacillus  enteritidis? 

13.  What  is  botulismus,  or  allantiasis? 

14.  What  is  ichthyosismus  ? 

15.  Can  botulismus  be  contracted  from  well-cooked  articles  of  food?    If  not, 
Why  not? 

16.  What  are  the  symptoms  of  botulismus  in  man? 

17.  Describe  the  morphologic  properties  of  the  Bacillus  botulinus. 

18.  Describe  its  cultural  characteristics  on  glucose  gelatin  plates. 

19.  Describe  the  spores  and  discuss  their  resistance. 

20.  What  is  the  optimum  temperature  of  the  Bacillus  botulinus? 

21.  Describe  its  pathogenic  properties  toward  laboratory  animals. 

22.  What  is  meant  by  a  pathogenic  or  toxigenic  saprophyte? 

23.  Discuss  the  resistance  of  the  botulinus  toxin. 

24.  What  are  its  features  of  similarity  with  the  tetanus  toxin? 

25.  What  measures  does  Van  Ermengem  recommend  to  prevent  botulismus  ? 


CHAPTER    XLIII. 

BACTERIA  OF  THE  NITROGEN  CYCLE— FERMENTATION  OF  UREA 
—NITRIFYING  AND  DENTRIFYING  ORGANISMS- 
FREE  NITROGEN  FIXATION. 

THE  importance  of  the  nitrogen  cycle  in  its  relation  to  the  main- 
tenance of  vegetable  and  animal  life  has  already  been  briefly  referred 
to  in  Chapter  II.  It  was  there  stated  that  bacteria  and  other  low 
vegetable  organisms  perform  a  most  important  function  of  this  cycle. 
The  subject  will  now  be  taken  up  in  greater  detail  in  the  discussion 
of  the  particular  bacteria  concerned  in  these  phenomena. 

Plants,  which  are  ultimately  necessary  for  the  preservation  of 
animal  life,  obtain  their  nitrogen  from  the  soil,  chiefly  in  the  form  of 
nitrates  (i.  e.,  salts  of  nitric  acid),  and  to  only  a  small  extent  in  the 
form  of  ammonia  salts.  As  the  amount  of  nitrates  in  the  soil  is  limited, 
it  would  soon  become  sterile  if  the  nitrates  were  not  replaced.  The 
nitrates  taken  up  by  the  higher  plants  are  changed  in  their  metabolism 
into  vegetable  proteids,  such  as  the  gluten  of  grasses  and  the  legumen 
of  leguminosse  (clover,  peas,  beans,  lentils,  etc.),  and  these'  in  turn 
when  taken  up  as  vegetable  food  are  changed  into  other  proteids  in 
the  animal  body.  Man  and  the  lower  animals  excrete  the  nitrogen 
waste  mainly  in  the  form  of  urea.  Lafar  states  that  mankind  excretes 
daily  about  37,500  tons  of  urea,  containing  seventeen  million  kilo- 
grams of  combined  nitrogen,  while  animals  certainly  excrete  a  much 
larger  daily  amount  of  this  nitrogenous  waste  product.  Urea  cannot 
be  directly  utilized  by  our  cultivated  plants  and  the  question,  therefore, 
arises,  How  does  this  animal  nitrogen-containing  waste  product 
become  available  again  as  a  source  of  nitrogen  for  the  building  up 
of  vegetable  proteids?  It  is  through  the  decomposition  of  the  urea 
by  the  action  of  bacteria  and  the  change  of  this  decomposition  product 
by  other  bacteria,  making  it,  finally,  again  utilizable  in  the  nutrition 
and  metabolism  of  plants.  By  decomposition  is  meant  the  breaking 
down  of  bodies  of  a  more  or  less  complex  chemical  composition  into 
simpler  compounds ;  in  other  words,  the  changing  of  a  complex  mole- 
cule into  two  or  more  simpler  molecules.  To  illustrate,  the  following 
examples  may  be  cited :  If  some  yellow  oxide  of  mercury  is  heated  in 
a  test-tube  it  is  decomposed  into  metallic  mercury  and  oxygen.  If  a 
proteid  is  exposed  to  the  action  of  pure  boiling  sulphuric  acid  in  a 
so-called  Kjeldahl  flask  this  very  complex  body  is  decomposed  into 
carbon  dioxide,  water,  ammonia,  and  other  simple  molecules.  What  is 
accomplished  with  the  proteid  by  artificial  chemical  manipulation  is 


458  FERMENTATION  OF  UREA 

done  under  natural  conditions,  though  much  more  slowly,  by  bacteria. 
Some  of  the  latter,  principally  strict  or  facultative  anaerobics,  break 
down  or  decompose  the  proteid  material  into  still  comparatively 
complex  molecules  with  the  production  of  a  fetid  smell.  This  process 
is  called  putrefaction.  Other  bacteria,  mostly  aerobics,  produce  a 
more  complete  decomposition  of  the  proteids  into  very  simple  com- 
pounds, like  carbon  dioxide,  water,  and  ammonia.  This  process  is 
known  as  decay.  There  is  no  generic  difference  between  the  processes 
of  putrefaction  and  decay,  but  only  one  of  degree.  Some  of  the  most 
common  bacteria  of  putrefaction  and  decay  have  already  been 
described,  such  as  the  Bacillus  proteus,  the  Bacillus  subtilis,  the 
Bacillus  mesentericus  vulgatus,  etc.  The  ammonia  derived  from 
decomposing  urea  and  proteids  may  be  formed  in  such  a  manner  as 
to  remain  in  the  soil,  or  it  may  escape  into  the  air.  from  which  it  is 
subsequently  washed  down  into  the  soil  with  the  rain.  Plants,  as 
stated,  can  utilize  ammonia  salts  only  to  a  slight  extent  for  providing 
for  their  nitrogen  requirements,  and,  hence,  nitrogen  in  this  form  is 
of  little  value  to  higher  vegetables  which  require  nitrogen  in  the  form 
of  nitrates.  Some  bacteria  have  the  power  to  ferment  urea  and 
change  it  into  salts  of  ammonia;  other  bacteria  possess  the  property 
of  oxidizing  ammonia  salts  into  nitrates;  this  process  is  called  nitri- 
fication. The  oxidation  of  ammonia  salts  into  nitrates,  however,  does 
not  occur  immediately,  but  through  the  intermediary  process  of  the 
formation  of  nitrites,  that  is,  salts  of  nitrous  acid,  which  represents  a 
lower  stage  of  oxidation  than  nitric  acid. 


FERMENTATION  OF  UREA. 

Quite  a  number  of  bacteria  possess  the  property  of  fermenting 
urea  and  decomposing  it  into  ammonia,  from  which  salts  are  formed 
when  the  opportunity  offers  for  the  base  to  unite  with  an  acid.  It  is 
probable  that  this  converting  power  depends  upon  an  enzyme  known 
as  urase.  Some  of  the  principal  urea-fermenting  bacteria  are  the 
following : 

Micrococcus  Urese. — This  is  a  globular  bacterium  from  0.8  to  1 
micron  in  diameter,  frequently  found  in  diplococcus  or  tetrad  form. 
According  to  Leube  it  forms  on  gelatin  plates  after  twenty-four 
hours,  white  cultures  of  the  size  of  a  millet  seed,  w^hich  have  a  mother- 
of-pearl  luster,  a  sharp  margin,  and  a  smooth  surface.  After  ten  days 
the  colonies  are  quite  large  and  resemble  somewhat  a  drop  of  stearin 
which  has  fallen  upon  and  solidified  on  a  surface.  The  growth  does 
not  liquefy  gelatin. 

Micrococcus  Ureae  Liquefaciens. — This  is  a  larger  organism  than  the 
preceding.  The  cocci  have  a  diameter  from  1.25  to  2  micra.  They 
appear  singly  or  in  chains  of  three  to  ten  individual  cocci.  On  gelatin 
plates  the  organism,  after  two  days,  forms  in  the  depth  small,  white 


NITRIFYING  BACTERIA  459 

points,  which  under  a  low  magnification  appear  dark  gray,  round, 
with  sharp  margins.  After  growing  up  toward  the  surface  the  colonies 
become  larger,  assume  a  yellowish-brown  color  and  a  dark  centre  and 
slowly  liquefy  the  medium.  In  gelatin  stick  cultures  a  white,  confluent 
mass  is  first  formed  along  the  stick  canal.  It  soon  leads  to  liquefaction 
and  the  latter  progresses  until  finally  one-half  or  more  of  the  medium 
has  become  liquefied,  while  the  bottom  is  covered  with  a  white, 
yellowish  sediment. 

Both  the  cocci  described  ferment  urea  when  present  in  the  culture 
media,  and  the  decomposition  continues  until  13  per  cent,  carbonate 
of  ammonium  has  been  formed.  The  optimum  temperature  of 
development  is  between  30°  to  33°  C.  The  best  artificial  culture 
medium,  according  to  von  Jaksch,  is  one  containing:  Urea,  3  gr. ; 
tartrate  of  sodium  and  potassium,  5  gr. ;  potassium  monophosphate, 
0.12  gr. ;  magnesium  sulphate,  0.06  gr.,  and  water  in  sufficient  quantity 
to  make  1000  c.c. 

Urobacillus  Pasteuri. — Miguel  has  isolated  from  air,  soil,  sewage, 
and  water  60  different  bacteria,  all  of  which  possess  the  property  of 
fermenting  urea.  Of  these  60  he  has  studied  17  species  more  par- 
ticularly, and  he  distinguishes  3  types,  namely,  the  urobacillus,  the 
urococcus,  and  the  urosarcina.  The  organism  which  has  the  greatest 
urea  fermenting  power  was  called  Urobacillus  Pasteuri  by  Miguel, 
who  found  it  frequently  in  sewage.  This  bacillus  can  split  up  140 
grams  of  urea  in  1000  c.c.  of  bouillon. 

NITRIFYING  BACTERIA. 

The  nitrifying  bacteria  possess  very  peculiar  properties  differing 
greatly  from  those  of  the  bacteria  considered  in  the  preceding  chapters. 
They  do  not  require  organic  material  for  their  growth  and  multi- 
plication, and,  in  fact,  do  not  grow  properly  in  its  presence  in  artificial 
culture  media.  The  latter  must  contain  simple  chemical  compounds 
only.  Nitrifying  bacteria  do  not  require  the  presence  of  light  in  their 
synthetic  metabolic  processes.  Nitrification,  however,  will  take  place 
in  the  soil  in  the  presence  of  small  amounts  of  organic  matter,  but 
any  larger  amount  will  stop  it  even  in  the  soil,  and  it  does  not  occur 
in  the  manure  as  first  existing  in  a  concentrated  form.  After  putre- 
faction and  decay  in  manure  has  decomposed  most  of  its  organic 
matter,  nitrification  can  occur.  The  process,  likewise,  does  not  take 
place  in  an  acid  medium,  and  for  this  reason  soils  that  have  become 
quite  acid  by  the  decomposition  of  a  large  amount  of  organic  matter 
must  first  be  neutralized  by  carbonate  of  lime  before  there  can  be 
any  progress  in  nitrification.  The  nitrifying  bacteria  have,  however, 
a  wide  range  of  temperature,  and  nitrification  occurs  under  otherwise 
favorable  conditions  between  37°  F.  to  110°  R ;  it  is  best  at  99°  F.,  and 
almost  ceases  at  122°  F.  Since  nitrification  is  a  process  of  oxidation, 
it  requires  the  presence  of  considerable  quantities  of  air,  and  the 


460  DENITRIFYING  BACTERIA 

more  broken  up  and  mingled  with  air  the  soil  the  better  the  process 
of  oxidation  of  the  ammonia  salts. 

The  chemical  action  of  these  bacteria  in  the  soil  has  been  known 
for  a  long  time,  but  the  greater  part  of  more  accurate  knowledge  is  due 
to  Winogradsky,  who  was  the  first  to  devise  methods  of  obtaining 
them  in  pure  cultures.  Winogradsky's  first  method  consisted  in  the 
use  of  a  silicon-jelly  (water-glass  jelly),  difficult  to  prepare.  The 
formulae  for  his  later  fluid  culture  media,  which  are  easier  to  prepare, 
were  given  in  Chapter  X.  The  pure  cultures  obtained  enabled 
Winogradsky  to  show  that  the  nitrifying  bacteria  consist  of  two 
groups,  one  oxidizes  ammonia  compounds  into  nitrites,  the  other 
group  changes  nitrites  into  nitrates. 

Nitrosomonas  and  Nitrosococcus. — These  are  the  bacteria  of  the  first 
group.  Their  oxidizing  action  takes  place  according  to  the  chemical 
formula  (NH4)2O  -f  3O2  =  N2O3  +  4H2O.  Nitrosomonas  Europea 
is  found  in  soil  in  Europe.  It  is  a  short  rod  1.2  to  1.8  micra  long, 
provided  with  a  short  flagellum,  and  lively  motile.  Nitrosomonas 
Javanica  was  isolated  from  soil  in  Batavia,  it  is  apparently  round 
and  coccus-like,  has  a  diameter  of  0.5  to  0.6  micron,  and  has  a  very 
long  flagellum  (up  to  30  micra).  Nitrosomonas  Japonica  and  N. 
Africana  are  like  the  European  variety.  Nitrosococcus  has  been 
found  in  South  America  and  Australia.  They  are  large  cocci,  1.5  to  1.7 
micra  in  diameter,  not  motile,  and  possess  no  flagella.  The  organisms 
of  this  group,  according  to  Winogradsky,  are  easily  perishable  when 
desiccated.  The  flagellate  nitrosomonas  in  young  cultures  swarm 
around  in  a  lively  manner  and  impart  to  the  medium  an  opalescent 
character;  later  they  sink  to  the  bottom,  unite  in  zoogleal  masses,  and 
form  a  grayish,  gelatinous,  cloudy  sediment.  The  best  method  to  recog- 
nize the  finer  details  of  the  structure  of  these  organisms  consists  in  treat- 
ing cover-glass  preparations  with  Gram's  iodine  solution.  All  varieties 
of  nitrosomonas  do  not  show  the  swarming  stages,  some  form  zoogleal 
masses  from  the  start,  and  remain  in  this  stage  permanently. 

Nitrobacteria. — These  form  nitrates  from  nitrites,  according  to  the 
formula  N2O3  +  2O  =  N2O5.  They  are  small,  oval,  or  pear-shaped 
bodies,  0.5  micron  long,  0.15  to  0.25  micron  wide.  They  form  in 
fluid  media  a  thin,  shiny  pellicle,  firmly  adherent  to  the  vessel  wall. 

The  two  groups  of  nitrifying  bacteria  in  soil  act  with  such  harmony 
that  it  is,  as  a  rule,  impossible  to  find  any  nitrites;  nitrates  alone  can 
be  discovered. 

DENITRIFYING  BACTERIA. 

While  the  nitrifying  bacteria  have  the  power  to  oxidize  nitrogen 
compounds  like  ammonia  to  lesser  or  higher  stages  of  oxidization, 
there  are  other  bacteria  which  have  -the  property  of  reducing  oxygen 
containing  nitrogen  compounds  into  lower  stages  of  oxidation  or  even 
,to  take  up  all  their  oxygen  and  set  the  nitrogen  free.  Such  organisms 
are  called  denitrifying  bacteria  in  a  general  sense.  Properly,  however, 


FIXATION  OF  FREE  NITROGEN  461 

this  term  should  be  reserved  exclusively  for  those  microorganisms 
which  are  able  to  set  nitrogen  free  from  nitrites  or  nitrates.  Many 
bacteria,  particularly  in  the  absence  of  free  oxygen,  under  anaerobic 
conditions,  can  obtain  their  oxygen  supply  by  a  reduction  of  nitrates 
into  nitrites,  and  among  these  is  the  Bacillus  coli  communis,  the 
typhoid  bacillus,  and  the  Bacillus  ramosus,  or  root  bacillus.  The 
bacilli  which  in  the  more  strict  sense  are  dentrifying  bacteria,  i.  e., 
those  which  can  carry  the  reduction  far  enough  along  to  the  setting 
free  of  nitrogen,  have  been  designated  as  Bacillus  denitrificans  a  and  /? 
(of  Gayon  and  Dupetit).  Aberson's  Bacillus  denitrificans  is  a  par- 
ticularly energetic  denitrifier  and  can  reduce  nitrates  contained  in  pure 
cultures  to  complete  liberation  of  all  of  the  nitrogen.  In  addition 
to  these  three  species,  others  of  the  group  have  been  described  by 
various  investigators.  These  bacteria  and  other  denitrifiers  may 
cause  great  loss  to  the  plant-available  nitrates  in  soil  under  faulty 
methods  of  fertilization,  as,  for  example,  particularly  when  nitrates 
and  fresh  manure  (especially  horse  manure)  are  distributed  simul- 
taneously to  the  soil. 

FIXATION  OF  FREE  NITROGEN. 

The  enormous  quantity  of  free  nitrogen  contained  in  the  atmosphere 
was  formerly  believed  to  be  entirely  useless  in  so  far  as  assimilation 
by  living  organisms  was  concerned.  Nitrogen  is  a  comparatively 
inert  gaseous  elementary  body,  unlike  oxygen,  which  easily  enters 
into  combination  with  a  variety  of  other  chemical  elements  and 
compounds.  It  has,  however,  been  known  for  some  time  that  certain 
plants  can  indirectly  derive  their  nitrogen  supply  from  the  atmosphere 
through  the  intervention  of  bacteria.  Most  plants,  if  brought  into  a 
nitrogen-free  soil,  wither  and  perish  after  a  short  time;  the  leguminosce, 
to  which  clovers,  peas,  beans,  lentils,  and  similar  plants  belong,  how- 
ever, can  thrive  even  in  a  nitrogen-free  soil.  They  develop  nodules 
from  the  size  of  a  pea  to  that  of  a  hazelnut  on  their  roots.  These 
root  nodules  of  the  leguminosse  contain  innumerable  bacteria  which 
have  united  with  the  higher  plant  in  a  symbiotic  community  and 
which  assimilate  free  nitrogen,  transforming  it  in  such  a  manner  that 
it  becomes  soluble  and  available  to  the  leguminous  plant  for  the 
preparation  of  the  required  vegetable  proteids.  The  exact  details  of 
this  chemical  transformation,  however,  are  not  fully  known,  but  the 
nitrogen  fixation  by  the  bacteria  of  the  root  nodules  of  the  leguminosse 
is  an  established  fact.  Wronin  was  the  first  investigator  who  observed 
that  the  root  nodules  contained  cells  filled  with  bacteria.  The  micro- 
scopic examination  of  the  nodule  shows  an  outer,  colorless  cortical  zone 
and  an  inner,  pale  red,  later  greenish-gray,  medullary  zone,  which  has 
rather  irregular  outlines  and  in  shape  somewhat  resembles  a  mulberry. 
This  inner  zone  is  composed  of  the  cells  which  contain  the  bacteria. 
Beyerinck,  in  1888,  first  isolated  such  bacteria  in  pure  cultures,  and 
he  named  the  organism  isolated  Bacillus  radicicola. 


462  FIXATION  OF  FREE  NITROGEN 

Cultural  Properties  and  Development  of  Nodule  Bacteria. — Prazmowski 
recommends  the  following  culture  medium:  An  infusion  is  prepared 
in  hot  water  from  leaves  of  leguminosse.  After  filtration  and  boiling, 
gelatin,  7  per  cent.;  asparagin,  J  per  cent.;  saccharose,  0.5  per  cent., 
are  added.  The  medium  is  standardized  so  that  it  contains  0.6  c.c. 
normal  acid  to  each  100  c.c  The  medium  is  kept  in  Petri  dishes 
which  are  inoculated  in  the  following  manner:  A  young  nodule  is 
first  washed  in  sterile  water,  afterward  placed  for  a  short  time  in 
strong  alcohol,  and  finally  washed  in  ether,  which  is  allowed  to 
evaporate.  When  dry  the  nodule  is  divided  with  a  sterile  knife  and 
the  juice  which  escapes  is  spread  with  a  platinum  loop  over  the 
surface  of  the  medium,  as  it  is  there  that  the  development  of  nodule 
bacteria  occurs  best.  Beyerinck  described  the  organism  so  obtained 
as  showing  two  morphologic  types.  One  is  a  rod  4  to  5  micra  long, 
1  micron  thick,  and  the  other  a  very  small  swarming  rod,  0.9  micron 
long  and  only  0.18  micron  thick.  The  small  bacilli  can  pass  the 
Chamberland  filter  and  they  wander  away  from  their  colonies  on  the 
soft  gelatin  plates  to  form  new  colonies  at  a  distance.  The  larger 
rods  are  by  no  means  regularly  cylindrical,  but  some  show  branched 
forms  shaped  like  a  Y.  The  Bacillus  radicicola  does  not  liquefy 
gelatin,  starch,  or  cellulose,  and  does  not  form  spores.  It  is  killed  at 
60°  to  70°  C.,  but  is  resistant  to  desiccation  or  freezing. 

Role  of  the  Nodule  Bacteria. — The  nodule  bacteria  of  leguminosse 
evidently  differ,  because  those  of  one  species  often  cannot  form 
nodules  in  another  species,  and,  as  a  rule,  can  infect  only  species  which 
are  very  nearly  allied.  For  instance,  bacteria  of  the  pea  can  form 
nodules  of  the  root  of  the  bean,  but  not  on  the  roots  of  different 
species  of  clover;  similarly,  the  bacteria  of  clover  cannot  infect  and 
enter  into  symbiotic  community  with  the  families  Vicia  (pea)  and 
Phaseolus  (bean).  Conn  (Agricultural  Bacteriology),  discussing  the 
role  of  these  microorganisms,  states:  "It  is  practically  certain  that 
nearly  all  soils  contain  bacteria  capable  of  living  in  symbiosis  with 
leguminous  plants.  Nearly  all  soils  except  extremely  sandy  soils,  that 
support  little  or  no  vegetation,  will  support  leguminous  plants  and 
develop  tubercles  on  their  roots.  One  can  scarcely  examine  the 
roots  of  legumes  anywhere  without  finding  tubercles,  a  fact  which 
shows  that  the  bacteria  in  question  are  very  widely  distributed  in 
nature.  But  are  the  bacteria  all  of  the  same  species?  A  very  large 
number  of  species  of  legumes  with  their  tubercles  can  grow  in  most,  if 
not  in  all,  soils.  Are  the  bacteria  that  form  tubercles  on  the  clover  the 
same  as  those  which  form  them  upon  the  pea,  or  is  there  a  different 
species  of  bacteria  for  the  different  species  of  legume?  It  would 
not  seem  probable  that  there  could  be  in  the  soil  a  different  variety 
of  bacteria  for  every  variety  of  legume,  but  rather  that  one  kind  of 
bacteria  can  grow  in  many  legumes.  But  the  facts  are  not  quite  so 
simple  as  this.  Not  all  species  of  legumes  are  capable  of  developing 
root  tubercles  equally  well  in  all  soils.  Some  soils  will  luxuriantly 


ROLE  OF  THE  NODULE  BACTERIA  463 

support  certain  species  of  beans,  peas,  or  clovers  producing  a  large 
crop,  developing  quantities  of  tubercles  and  fixing  an  abundance  of 
nitrogen,  while  the  same  soil  will  not  support  other  species  of  legumes 
with  equal  readiness.  ...  It  certainly  means  that  different 
species  of  legumes  demand  different  varieties  of  tubercle  bacteria. 
Whether  these  different  varieties  are  distinct  species  is,  of  course, 
a  fruitless  question,  inasmuch  as  we  do  not  know  what  we  mean 
by  a  species  among  bacteria.  But  it  is  of  importance  to  know  whether 
these  types  are  quite  distinct  or  whether  they  are  simply  physiological 
varieties  of  the  same  general  species.  If  the  former  is  true  we  should 
expect  them  to  remain  distinct,  but  if  the  latter  is  true,  we  might 
expect  the  soil  bacteria  to  be  capable  of  adaptation  by  cultivation 
to  different  legumes.  On  the  whole,  the  evidence  is  decidedly  in 
favor  of  the  latter  view  and  indicates  that  the  different  tubercle 
bacteria  are  probably  all  one  general  species,  but  that  under  different 
conditions  they  assume  slightly  different  physiological  relations. 
They  can  accommodate  themselves  to  grow  in  one  or  another  legume, 
and  having  become  especially  adapted  for  one  species,  but  allowed 
to  develop  in  the  soil  in  which  the  latter  plants  are  growing,  they 
will  adapt  themselves  in  time  to  the  new  plant.  In  other  words, 
experiments  indicate  that  there  is  probably  one  species  of  tubercle 
bacteria,  and  that  this  species  assumes  different  physiological  char- 
acters under  the  influence  of  the  different  conditions  in  which  it 
grows." 

The  Bacillus  or  Bacterium  radicicola,  according  to  Prazmowski, 
penetrates  into  the  epidermis  cells  of  the  root  hairs  of  leguminosse 
and  develops  a  colony  which  surrounds  itself  with  a  tough  membrane. 
From  the  point  of  entrance  a  sac  filled  with  bacteria  is  then  formed. 
It  grows  toward  the  cortical  cells  and  penetrates  into  the  interior  of 
the  root  hair,  where  it  stimulates  the  cells  to  proliferation.  The  sacs, 
also  called  the  infectious  filaments,  are  not  part  of  the  leguminous 
plant,  but  are  derived  from  the  gelatinous  (zoogleal)  substance  of 
the  bacteria.  The  different  parts  which  constitute  the  infected 
cells  can  be  demonstrated  by  a  mixed  watery  solution  of  fuchsin 
and  gentian  violet  in  1  per  cent,  acetic  acid.  Sections  of  the  nodules 
or  tubercles  stained  with  this  solution  show  the  plasmatic  contents 
and  the  membrane  of  the  leguminosa  cells  blue,  the  bacteria  red, 
and  their  common  zoogleal  envelope  and  membrane  unstained.  The 
entire  mass  of  the  tubercles,  composed  of  the  infected  leguminosa 
cells  and  the  infecting  bacteria,  has  been  called  the  bacterioid  tissue. 
The  bacteria  themselves,  after  infecting  the  higher  plant  cells,  undergo 
changes  and  degenerate  into  involution  forms  which  have  been 
called  bacterioids  (this  means  bacteria-like  forms).  These  are  quite 
pleomorphous,  have  branches  and  sub-branches,  and  often  form  a 
more  or  less  regular  complete  reticulum,  or  network.  The  nitrogen 
content  of  the  dry  substance  of  these  root  nodules  of  leguminosse 
is  very  high.  It  was  estimated  by  Stoklasa,  at  the  time  of  flowering 


464  FIXATION  OF  FREE  NITROGEN 

of  the  plants,  at  5.2  per  cent. ;  at  the  time  when  the  fruit  begins  to 
form,  at  2.6  per  cent.;  and  after  the  fruit  had  become  ripe,  at  1.7  per 
cent.  The  exact  manner  in  which  the  nitrogen  is  taken  up  by  the 
plant  from  the  root  tubercles  is  not  known.  Frank  found  even  higher 
percentages — namely,  6.94  and  7.44  per  cent,  of  nitrogen — and  since 
the  latter  is  present  in  the  form  of  proteids  it  means  a  percentage  of 
43.4  and  46.5  of  dry  proteid  substance. 

Clover  and  other  leguminosse  are  now  frequently  used  as  so-called 
green  manure  for  improving  impoverished  soil.  These  leguminosse 
which  can  obtain  their  nitrogen  supply  with  the  assistance  of  the 
nitrogen-fixing  bacteria  from  the  air  are  planted  in  the  nitrogen-poor 
oil.  They  are  allowed  to  grow,  and  instead  of  being  harvested  are 
plowed  under.  The  chemical  and  biological  facts  of  nitrogen  absorp- 
tion have  been  known  for  a  few  decades  only,  but  the  ancient  Romans 
had  already  noted  the  fact  that  impoverished  soil  could  be  improved 
by  the  planting  of  clover. 

Clostridium  Pasteurianum. — In  addition  to  those  bacteria  which  have 
the  power  of  nitrogen  fixation  in  symbiotic  community  with  legumin- 
osse,  there  are  other  bacteria  which  exhibit  the  same  power  in  soil 
alone  and  without  being  in  symbiosis  with  other  higher  organisms. 
The  investigations  of  Winogradski  have  demonstrated  this.  He 
described  a  nitrogen-fixing  bacterium  under  the  name  of  Clostridium 
Pasteurianum.  It  is  a  rod  about  5  micra  long,  1.2  micra  wide,  which 
produces  end  spores,  and  in  doing  so  assumes  the  clostridium  shape. 
At  the  same  time  it  forms  in  its  interior  (but  not  at  the  poles)  sub- 
stances which  are  stained  deep  black  blue  with  iodin  solution.  The 
mature  spores  escape  in  the  longitudinal  axis  of  the  organism.  The 
organism  belongs  to  the  group  of  butyric-acid  bacteria,  to  which  the 
bacilli  of  black-leg  and  malignant  edema  also  belong.  The  Clos- 
tridium Pasteurianum  is  a  strict  anaerobe  like  most  members  of  this 
group.  It  forms  butyric  and  also  acetic  acid  in  the  presence  of 
carbohydrates,  which  are  used  as  a  source  of  energy;  it  can  fix  free 
nitrogen  from  the  air.  The  organism  does  not  grow  on  the  general 
artificial  culture  media,  but  on  potatoes.  As  the  medium  best  adapted 
for  its  growth  the  following  is  recommended : 

Phosphate  of  potassium  (K3PO4) 1.0 

Sulphate  of  magnesium 0.5 

Chloride  of  sodium, 

Sulphate  of  iron, 

Sulphate  of  manganese       ....      1     .     .....    each  0.01  to    0.02 

Carbonate  of  calcium,  enough  to  neutralize. 

Glucose 20. 00  to  40.0 

Water,  enough  to  make 1000 . 00  c.c. 

As  the  Clostridium  Pasteurianum  is  strictly  anaerobic  it  can  develop 
in  soil  only  in  the  presence  of  aerobic  bacteria,  which  use  up  the  oxygen. 

Azotobacters. — A  group  of  aerobic  bacteria  able  to  fix  nitrogen  has 
been  discovered  by  Beyerinck.  He  has  named  them  Azotobacter. 
These  organisms  are  oval  bacteria,  4  to  6  micra  long;  they  are 


QUESTIONS  465 

motile  and  possess  flagella.  Spore  formation  has  not  been  observed. 
The  Azotobacter  agilis  is  more  lively  motile  than  the  Azotobacter 
chroococcum. 

QUESTIONS. 

1.  What  is  meant  by  the  nitrogen  cycle?    Why  is  it  necessary  to  preserve 
animal  and  vegetable  life  on  our  planet? 

2.  For  what  purpose  do  plants  and  animals  need  nitrogen? 

3.  In  what  form  can  plants  utilize  nitrogen  for  their  nitrogen  metabolism? 

4.  In  what  form  do  man  and  the  lower  animals  mainly  excrete  the  waste 
nitrogen  ? 

5.  What  is  meant  by  a  metabolic  waste  product? 

6.  What  becomes  of  urea  excreted  by  animals? 

7.  What  is  meant  by  decomposition  of  complex  chemical  compounds? 

8.  Give  some  examples. 

9.  What  is  the  difference  between  putrefaction  and  decay? 
10.  Name  some  putrefactive  bacteria? 

-11.  What  is  meant  by  nitrifying  bacteria? 

12.  Describe  some  of  their  peculiar  biologic  properties. 

13.  Under  what  conditions  will  nitrification,  brought  about  by  bacteria,  go 
on  in  the  soil? 

14.  What  microorganisms  have  the  power  to  decompose  urea?    Name  some 
of  the  most  important  ones. 

15.  Describe  the  Micrococcus  urese. 

16.  Describe  the  Micrococcus  urese  liquefaciens. 

17.  Describe  the  Urobacillus  Pasteuri. 

18.  What  is  the  difference  in  action  between  the  two  groups  of  nitrifying 
bacteria? 

19.  Describe  the  different  species  of  nitrosomas. 

20.  Describe  a  species  of  nitrosococcus. 

21.  In  what  fluid  are  they  best  examined  microscopically? 

22.  Describe  the  morphology  and  the  growth  of  nitrobacteria. 

23.  What  is  meant  by  denitrification  ? 

24.  Name  some  bacteria  which  in  the  absence  of  free   oxygen  can  act  as 
denitrifiers. 

25.  Under  what  circumstances  can  these  bacteria  produce  great  loss  of  nitrates 
in  improper  fertilization  of  the  soil? 

26.  What  plants  have  the  power  to  utilize  nitrogen  from  the  atmosphere  for 
their  metabolism? 

27.  What  are  the  root  tubercles  of  leguminosae? 

28.  What  is  the  name  of  the  bacillus  contained  in  the  root  tubercles?    What 
is  its  physiologic  function  ? 

29.  What  is  the  best  culture  medium  for  this  bacillus?    How  can  we  obtain 
pure  cultures  of  the  Bacillus  radicicola? 

30.  Describe  the  morphology  of  this  bacillus. 

31.  Discuss  the  question  whether  or  not  there  are  a  number  of  species  of  the 
Bacillus  radicicola. 

32.  Describe  how  this  organism  penetrates  into  the  root  hair  and  what  changes 
it  produces. 

33.  What  is  meant  by  bacterioid  tissue?    Describe  it  in  detail. 

34.  Discuss  the  nitrogen  content  of  root  tubercles  at  various  periods  of  the 
plant's  life. 

35.  What  is  meant  by  green  manuring?    What  is  its  object  and  effect? 

36.  Name  some  bacteria  which  are  nitrogen  fixers  but  not  in  symbiotic  com- 
munity with  higher  plants. 

37.  Describe  the  morphologic  and  biologic  properties  of  Clostridium  Pasteuri- 
anum. 

38.  What  kind  of  organism  is  azotobacter? 


30 


CHAPTER    XLIV. 

ACETIC-ACID  BACTERIA. 

NITRIFICATION  consists  in  an  oxidation  of  nitrogen,  hence  the 
nitrifying  bacteria  are  oxidizing  microorganisms.  Bacterial  oxida- 
tions are  not  confined  to  changes  in  the  soil  which  are  of  the  greatest 
significance  to  agriculture,  but  they  play  an  important  role  in  nature  in 
the  cycle  of  certain  elements  and  in  certain  industries.  One  of  these  is 
the  essential  chemical  process  in  the  manufacture  of  vinegar,  consist- 
ing in  the  conversion  of  alcohol  into  acetic  acid.  Alcohol  formation 
from  sugar  is  generally  due  to  yeast  cells  (blastomycetes  or  budding 
fungi),  and  the  conversion  of  liquids,  such  as  beer,  wine,  or  cider, 
containing  alcohol  into  vinegar,  that  is,  into  a  fluid  containing  acetic 
acid,  is  due  to  bacterial  microorganisms.  When  alcohol  containing 
liquids  in  open  vessels  and  exposed  to  the  air  are  changed  into 
vinegar  a  tenacious  slimy  membrane  is  formed  on  the  surface.  It 
was  noted  long  ago  that  pieces  of  this  membrane  rapidly  changed 
alcoholic  liquids  into  vinegar  in  the  presence  of  free  air;  the  membrane, 
therefore,  received  the  name  mother  of  vinegar  (mere  du  vinaigre, 
French;  Essigmiitter,  German).  Persoon,  in  1822,  examined  such 
membranes  in  various  fermentative  processes,  and  he  called  them 
mycoderma,  which  means  a  fungus,  or  slimy  skin  or  membrane,  but 
he  did  not  believe  in  any  causal  connection  between  them  and  the 
fermentative  process.  Kuetzing,  a  German  botanist,  however,  in  1832, 
declared  that  the  mother  of  vinegar  consisted  of  exceedingly  small, 
punctate  algae,  and  that  the  conversion  of  alcohol  into  acetic  acid  was 
due  to  their  metabolism.  Kuetzing's  claims  never  attracted  much 
attention,  and  had  long  been  forgotten,  when  more  than  forty  years 
later  Pasteur,  in  his  studies  on  fermentations,  again  maintained  that 
the  conversion  of  alcohol  into  acetic  acid  was  due  to  the  life  activity 
of  microorganisms  which  he  called  Mycoderma  aceti.  Pasteur,  how- 
ever, did  not  exhaustively  study  the  morphology  of  these  vinegar 
organisms;  this  was  later  done  by  Hansen,  who  distinguished  three 
different  species  of  bacteria  as  the  cause  of  the  acetic-acid  formation 
from  alcohol.  These  he  named  Bacterium  aceti,  Bacterium  Pasteuri- 
anum,  and  Bacterium  Kuetzingianum.  The  three  species  are  found 
in  beer  which  is  not  very  rich  in  alcohol  and  which  is  undergoing 
acetic-acid  fermentation.  Bacterium  aceti  forms  a  moist,  slimy, 
smooth  pellicle  with  fine  lines;  Bacterium  Pasteurianum,  a  rather 
dry  pellicle;  and  Bacterium  Kuetzingianum  one  which  resembles  that 
of  Bacterium  aceti,  but  is  much  thicker,  elevated,  and  reaches  up  along 


ACETIC-ACID  BACTERIA  467 

the  sides  of  the  vessel.  The  pellicles  formed  are  zoogleal  masses 
in  which  the  individual  bacteria  are  held  together  by  a  plasmatic, 
gelatinous  substance.  In  the  case  of  Bacterium  aceti  the  latter  is 
not  stained  by  iodine  solution,  while  in  the  other  two  vinegar  bacilli 
it  is  stained  blue.  The  bacterial  protoplasm  proper  of  all  three 
species  stain  yellow  with  iodin  solution.  Another  acetic-acid  bac- 
terium, commonly  known  in  England  as  vinegar  plant,  has  been 
described  by  Brown  as  the  Bacterium  xylinum.  It  forms  tough, 
leathery,  thick  zoogleal  masses.  The  three  species  of  Hansen  also 
show  marked  differences  in  pure  cultures  on  solid  media  (wort 
gelatin).  Bacterium  aceti  forms  very  delicate,  rosette-like  colonies; 
Bacterium  Pasteurianum  develops  colonies  with  a  smooth,  round 
periphery  and  folded  surface,  and  Bacterium  Kuetzingianum  shows 
smooth  colonies  without  any  surface  folds.  All  three  types  are  short, 
rather  thick  bacilli,  but  they  exhibit  considerable  pleomorphism 
under  various  conditions.  Their  optimum  temperature  of  growth 
lies  between  34°  and  42°  C.,  and  they  cease  multiplication  between 
5°  and  7°  C.  When  raised  at  a  temperature  of  34°  C.  the  Bacterium 
Pasteurianum  forms  chains  of  bacilli  which  are  about  2  micra  long 
and  1  micron  thick.  If  these  are  transferred  to  a  fresh  medium  kept 
at  40.5°  C.,  long  filaments  are  formed  in  which  no  dividing  lines  can 
be  seen,  and  which  reach  a  size  of  from  40  to  200  micra.  If  these 
are  again  transplanted  to  a  fresh  medium  kept  at  34°  C.,  globular  or 
elliptical  swellings  are  formed  in  the  threads,  and  these  later  break  up 
into  short  rods  and  pear-shaped  or  globular  cells,  sometimes  measuring 
10  micra  in  diameter.  The  other  two  acetic-acid  bacilli  of  Hansen 
under  similar  conditions  likewise  show  much  pleomorphism  and  even 
develop  branched  forms. 

The  conversion  of  alcohol  into  acetic  acid  is  a  process  of  oxidation, 
represented  by  the  formula: 

C2H60      +     02     =     C2H402     +     H20. 

Alcohol.  Oxygen:       Acetic  acid.  Water. 

In  the  manufacture  of  vinegar  from  alcoholic  liquids,  such  as  wine, 
beer,  and  cider,  provision  must  always  be  made  that  the  air  has  free 
access  to  the  fermenting  fluid.  This  is  accomplished  by  either 
allowing  air  to  enter  the  barrels  from  one  side  and  to  escape  from 
another,  or  by  leading  the  fermenting  fluid  through  barrels,  vats, 
or  tubes  containing  masses  of  wood  shavings.  This  arrangement 
spreads  the  fluid  over  a  large  surface  and  allows  it  to  mix  thoroughly 
with  air.  Pure  cultures  of  acetic-acid  bacteria  are  not  yet  generally 
used  in  the  manufacture  of  vinegar,  though  it  appears  that  there 
would  be  considerable  advantage  in  such  a  procedure.  The  use  of 
pure  cultures  of  yeasts,  as  is  well  known,  has  done  very  much  in 
improving  the  quality  of  beer  and  preventing  losses  from  the  develop- 
ment of  undesirable,  so-called  wild  yeasts 


468  ACETIC  ACID  BACTERIA 


QUESTIONS. 

1.  What  kind  of  chemical  process  is  the  conversion  of  alcoholic  liquids  into 
vinegar? 

2.  What  is  vinegar? 

3.  What  generally  causes  the  conversion  of  glucose  into  alcohol  and  carbon 
dioxide  ? 

4.  What  causes  the  conversion  of  alcohol  into  acetic  acid  and  water? 

5.  What  is  meant  by  the  mother  of  vinegar? 

6.  What  was  the  first  scientific  name  proposed  for  mother  of  vinegar? 

7.  Name  some  acetic-acid  microorganisms. 

8.  Describe  their  morphology  and  their  cultural  characters. 

9.  How  do  the  zooglea  of  different  acetic-acid  bacteria  behave  toward  iodin 
solution  ? 

10.  What  is  the  chemical  equation  for  the  conversion  of  alcohol  into  acetic 
acid  and  water?    What  kind  of  a  process  is  it? 

11.  What  is  the  general  arrangement  of  the  manufacture  of  vinegar? 


CHAPTEE    XLV. 

THE  BACTERIOLOGY    AND  THE    BACTERIOLOGIC    EXAMINATION 
OF  MILK1— GENERAL   INTRODUCTORY    CONSIDERATIONS— 
THE    CHANGE    OF    LACTOSE    INTO    LACTIC     ACID— 
LACTIC-ACID  BACTERIA— ANAEROBIC  BUTYRIC- 
ACID  FORMERS— PEPTONIZING  BACTERIA 
—ALCOHOLIC  FERMENTATION 
OF   MILK. 

GENERAL  INTRODUCTORY  CONSIDERATIONS. 

WHILE  milk  is  an  excellent  culture  medium  for  many  bacteria, 
there  are  also  many  which  do  not  find  in  milk  conditions  favorable 
to  their  growth.  It  is  practically  impossible  to  obtain  milk  from 
animals  in  an  absolutely  germ-free  or  sterile  condition.  This  statement 
can  perhaps  best  be  explained  by  the  analogy  of  surgeons7  attempts 
to  obtain  completely  germ-free  hands  before  an  operation.  Such 
endeavors  were  begun  soon  after  the  knowledge  of  pyogenic  wound 
infection  microorganisms  and  their  ubiquitous  nature  became  estab- 
lished and  have  been  continued  for  several  decades.  A  vast  litera- 
ture upon  this  subject  has  accumulated,  but  notwithstanding  trials 
continued  for  many  minutes  up  to  one-half  hour  and  more  it  is  now 
conceded  to  be  impossible  to  sterilize  the  human  hands.  While 
washing  and  scrubbing  with  disinfectants,  such  as  soap  and  water, 
dilute  alcohol  and  corrosive  sublimate  solution  may  render  the  surface 
of  the  hands  temporarily  germ  free,  the  cracks  and  recesses  of  the 
skin  and  the  ducts  of  the  sweat  and  sebaceous  glands  remain  infected 
with  bacteria,  which  when  the  hands  are  used  and  when  perspiration 
occurs  soon  find  their  way  to  the  surface. 

This  being  the  case  it  would  be  unreasonable  to  expect  that  the 
external  surface  of  the  udder  and  the  hands  of  the  milker  could  be 
so  sterilized  as  to  render  them  entirely  free  from  bacteria.  Even 
were  this  possible  the  bacteria  always  occurring  in  the  milk-ducts  still 
remain,  and  they  would  find  their  way  into  the  milk  during  milking. 
For  this  reason  it  may  be  stated  that  milk  after  collection  is  practically 

1  The  bacteriology  and  hygiene  of  milk  have  been  treated  as  fully  as  is  consistent  in  a  text- 
book on  bacteriology.  For  a  more  extensive  consideration  the  reader  is  referred  to  the  following 
works:  Sommerfeld,  Handbuch  der  Milchkunde,  Wiesbaden,  1909;  Jensen,  Essentials  of 
Milk  Hygiene,  translated  by  Pearson,  Philadelphia,  1909;  Conn,  Bacteria  in  Milk,  Phila- 
delphia, 1903;  Swithinbank  and  Newman,  Bacteriology  of  Milk,  New  York,  1903;  Milk  and 
its  Relation  to  Public  Health,  Public  Health  and  Marine  Hospital  Service  of  the  United 
States,  Washington,  Bulletin  No.  56;  Russel,  Outlines  of  Dairy  Bacteriology;  Ward,  Pure 
Milk  and  the  Public  Health;  Winslow,  Clean  Milk. 


470  THE  CHANGE  OF  LACTOSE  INTO  LACTIC  ACID 

never  sterile,  nor  did  nature  attempt  or  intend  to  furnish  to  the  young 
animal  or  infant  an  absolutely  sterile  germ-free  food  supply.  The 
milk  as  drawn  by  suction  from  the  milk  glands  becomes  mixed  with 
bacteria  from  the  ducts,  and  the  skin  and  the  bacterial  contents  are 
further  much  augmented,  before  the  stomach  is  reached,  by  the 
admixture  with  the  secretions  of  the  mouth,  which  contain  very 
numerous  bacteria. 

The  general  statement  that  absolutely  sterile  milk  can  never  be 
obtained  refers,  of  course,  to  the  practical  collection  of  the  fluid  from 
the  cow.  A  trained  bacteriologist,  after  the  liberal  removal  of  the 
foremilk  and  a  larger  amount  of  milk  flowing  subsequently,  can 
collect  a  few  cubic  centimeters  of  bacteria-free  milk  in  a  sterile  test- 
tube,  as  has  been  shown  in  Bergey's  extensive  bacterial  milk  analyses. 

What  has  been  said,  however,  must  not  be  taken  to  imply  that 
efforts  should  not  be  made  to  obtain  milk  in  such  a  way  as  to  keep 
its  bacterial  contents  as  low  as  possible.  This  can  best  be  accom- 
plished by  using  the  greatest  care  in  collecting  the  milk,  by  cleaning 
the  udder,  cleansing  the  milker's  hands,  tying  the  tail  of  the  cow 
during  milking,  receiving  the  milk  into  a  clean  (if  possible  sterile) 
vessel  washed  out  with  boiling  hot  water,  protecting  the  lacteal  fluid 
afterward  from  contamination  with  dust  and  dirt,  and  cooling  it 
rapidly  and  keeping  it  cool  to  prevent  subsequent  bacterial  growth 
and  multiplication. 

Numerically,  most  bacteria  in  contaminated  milk  are  derived  from 
fecal  matter  of  the  cow.  \\iithrich  and  Freudenreich  have  ascer- 
tained that  the  feces  of  the  cow  contain  about  375,000,000  bacteria 
per  gram  of  moist  substance,  hence  it  is  of  the  greatest  importance 
in  collecting  milk  to  guard  against  contamination  with  cow's  dung 
and  against  the  dust  and  dirt  derived  from  it.  In  practice,  however, 
it  has  been  recognized  that  a  certain  amount  of  dust  and  dirt  con- 
tamination is  unavoidable,  and  various  bacteria  from  those  sources, 
in  addition  to  those  habitually  present  in  the  milk-ducts,  must  be 
expected  to  be  found,  no  matter  how  clean  and  ideal  the  environments 
of  the  milch  cow's  stable.  Renk  and  others  have  elaborated  methods 
to  ascertain  the  amount  of  dirt  which  can  be  removed  by  sedimen- 
tation from  milk,  and  which  can  subsequently  by  exact  chemical 
methods  be  dried  and  weighed.  European  authorities  appear  to  agree 
that  milk  collected  by  cleanly  methods  should  contain  less  than  10 
milligrams  of  dry  dirt  per  liter,  i.  e.,  less  than  one  part  to  100,000 
parts  of  milk;  but  otherwise  good  market  milk  often  contains  an 
amount  many  times  in  excess  of  this  standard. 

THE  CHANGE  OF  LACTOSE  INTO  LACTIC  ACID. 

Even  when  milk  is  collected  in  a  very  clean  and  careful  manner 
it  will  undergo  certain  changes  and  generally  become  sour  unless 
permanently  kept  at  a  temperature  very  near  the  freezing  point  of 


THE  LACTIC-ACID  BACTERIA  471 

water.  This  souring  of  milk  is  due  to  the  accumulation  of  lactic  acid, 
which  is  formed  from  the  lactose,  or  milk  sugar,  of  the  milk.  This 
change  is  brought  about  by  a  great  variety  of  bacteria,  which,  broadly 
speaking,  are  always  present  in  milk,  and  which,  in  their  relation  to 
milk,  are  known  under  the  collective  name  of  lactic-acid  bacteria. 
The  grouping  is  entirely  arbitrary  and  artificial,  because  the  organ- 
isms belong  to  various  types  and  have  in  common  only  the  property 
of  splitting  up  lactose  and  forming  from  it  lactic  acid. 

The  change  of  lactose  into  lactic  acid  is  chemically  a  process  of 
hydrolysis,  i.  e.,  a  chemical  change  in  which  water  is  added  to  a 
molecule,  and  this  molecule  subsequently  becomes  split  up  into  other 
compounds.  The  change  is  probably  due  to  a  so-called  soluble 
ferment  or  enzyme,  secreted  by  the  lactic  acid  bacteria  or  contained 
in  their  bodies,  where  it  may  act  upon  the  lactose  which  diffuses  into 
the  substance  of  the  bacterium  by  osmotic  processes.  The  chemistry 
of  the  process  is  expressed  by  the  following  formula  : 


+     H20     =       2C6H120B       =    4C3H603 

Lactose        +     Water      =      1  Glucose  and      =       4  Lactic 
1  Galactose  acid 

In  order  to  understand  the  names  given  to  some  of  the  bacteria  of 
the  lactic-acid  group  it  is  necessary  to  know  that  lactic  acid  is  a 
stereoisomeric  body.  This  term  means  a  body  or  chemical  substance 
existing  in  nature  in  two  forms  of  crystallization,  which  bear  the 
relation  to  each  other  of  a  physical  object  to  its  image  in  a  plane 
mirror  and  which,  when  in  solution,  will  behave  in  the  following 
peculiar  manner  toward  rays  of  polarized  light:  One  form  of  crystals, 
or  bodies  in  solution,  will  deflect  or  deviate  the  polarized  rays  of  light 
from  their  straight  path  toward  the  right  side,  and  these  are  called 
dextrogyr;  the  other  form  will  deviate  or  deflect  the  polarized  rays 
of  light  from  their  straight  path  toward  the  left  side,  and  these  are 
called  sinistrogyr,  or  levogyr.  By  a  mixture  of  these  two  forms  a 
solution  may  be  obtained  which  will  deflect  the  polarized  light, 
neither  to  the  right  nor  to  the  left  side. 


THE  LACTIC-ACID  BACTERIA. 

The  bacteria  most  commonly  found  in  milk  and  producing  in  it 
the  most  rapid  and  obvious  changes  are  those  which  possess  the 
power  to  ferment  milk  sugar  (lactose)  and  form  from  it  lactic  acid. 
They  are  known  collectively  as  the  lactic-acid  bacteria  and  occur 
very  widespread  in  nature.  They  have  been  found  in  hay  and  straw 
(Leichmann,  Gruber,  and  others),  also  in  the  dust  in  barns  and  other 
places,  on  ordinary  grasses  and  cereals,  and  other  cultivated  plants. 
Beyerinck  found  them  in  the  feces  of  man  and  animals,  and  Barthel 
has  found  them  practically  wherever  man,  animals,  and  cultivated 


472  THE  LACTIC-ACID  BACTERIA 

plants  are  found.  It  has  already  been  stated  that  numerically  the 
most  important  source  of  bacteria  in  milk  is  cow's  dung,  in  which 
enormous  numbers  of  bacilli  of  the  colon-aerogenes  group  are  found. 
These  are  lactic-acid  formers.  Such  bacteria,  however,  may  also 
enter  the  milk  from  other  sources.  It  was  pointed  out  in  the  chapter 
on  the  Bacilli  of  the  Colon  Group  that  a  number  of  investigators  look 
upon  the  Bacillus  coli  communis  as  a  ubiquitous  organism.  If  this 
is  the  case  its  presence  in  milk  cannot  be  taken  as  an  absolute  indi- 
cation of  fecal  contamination.  Rodgers  and  Ayers,  in  Circular  135  of 
the  Bureau  of  Animal  Industry,  state  that  in  some  middle  Western 
States  organisms  of  the  colon  aerogenes  group  are  commonly  found 
on  grass,  grain,  and  in  slough  holes,  ,and  that,  therefore,  these  lactic- 
acid  and  gas  formers  may  not  be  derived  from  a  fecal  source  when 
cows  are  milked  in  open  fields.  The  discovery  that  the  lactic-acid 
fermentation  of  milk  is  due  to  bacteria  was  first  made  by  Pasteur  and 
later  confirmed  by  Lister.  Hiippe  gave  the  first  description  of  a 
bacterium  of  this  type  and  named  it  Bacillus  acidi  lactici,  and  orig- 
inally believed  that  the  latter  was  the  only  microbe  which  caused 
lactic-acid  fermentation  of  milk.  It  was,  however,  soon  shown  by  a 
number  of  investigators  (Grotenfeld,  Marpmann,  Conn,  Weigmann, 
and  others)  that  a  great  variety  of  bacteria  occur  in  milk  which  possess 
the  power  to  ferment  lactose.  Some  of  them  produce  a  dextrogyr, 
others  a  sinistrogyr  lactic  acid.  The  former  type  was  found  first. 
To  the  second  class  belongs  Leichmann's  Bacillus  lactis  acidi  and 
Micrococcus  acidi  levolactici  and  Kozai's  Bacillus  acidi  levolactici. 

Classification. — The  lactic-acid  bacteria  are  now,  according  to 
Loehnis,  as  quoted  by  Weigmann,  divided  into  four  groups,  namely: 

Group  I. — Plump,  Gram-negative,  gas-forming  rods  designated  as 
Bacterium  pneumonia?  of  Friedlander  or  Bacterium  acidi  lactici  of 
Hiippe. 

Group  II. — Elongated,  oval,  or  lancet-shaped,  Gram-positive  strep- 
tococci, growing  anaerobically  and  forming  very  little  gas  designated 
as  Streptococcus  pyogenes  or,  better,  as  Streptococcus  Guentheri. 
This  group  contains  the  most  important  lactic-acid  formers  occurring 
in  milk. 

Group  III. — Long,  slender,  Gram-positive  bacilli,  growing  better 
anaerobically  and  forming  little  gas,  designated  as  Bacterium 
caucasicum  or  Bacterium  casei. 

Group  IV. — Gram-positive  staphylococci,  aerobic,  forming  no  gas, 
generally  liquefying  gelatin  and  designated  as  Micrococcus  pyogenes 
or  Micrococcus  lactis  acidi. 

Bacteria  of  Group  I. — The  bacteria  of  the  first  group  are  generally 
short  rods,  1  to  1^  micra  long  and  0.75  to  1  micron  thick.  They 
occur  singly  or  in  groups  of  two,  and  also  in  the  form  of  longer  chains 
They  are  not  motile  and  do  not  form  spores,  generally  they  are 
Gram  negative.  They  are  aerobic  and  facultative  anaerobic.  As  a 
rule,  they  coagulate  milk  in  from  one  to  two  days,  sometimes  later, 


THE  LACTIC-ACID  BACTERIA  473 

and  occasionally  coagulation  does  not  occur  at  all,  but  the  milk 
becomes  thready  or  slimy.  The  lactic  acid  formed  is  generally  of 
the  sinistrogyr  variety.  These  bacteria  grow  in  milk  between  15°  and 
45°  C.,  and  best  between  30°  and  40°  C.  They  often  impart  a  dis- 
agreeable taste  to  milk  and  occasionally  milk  containing  them  in 
very  large  numbers  causes  vomiting.  These  bacteria  ferment  other 
sugars,  in  addition  to  lactose,  and  then  form,  besides  lactic  acid,  also 
succinic,  acetic,  and  some  formic  acid;  they  also  sometimes  form 
alcohol  and  carbon  dioxide  and  hydrogen.  The  several  types  in  this 
first  group  are  represented  as  follows: 

Type  1. — Gas  formers:  Bacillus  acidi  lactici  Hiippe,  Bacillus 
lactis  aerogenes  Escherich,  Grotenfeld's  Bacillus  acidi  lactici,  the 
fan-bacillus  of  Clauss,  and  Lustig's  typhoid-like  bacillus. 

Type  2. — These  coagulate  milk  but  do  not  form  gas:  Bacterium 
Hmbatum  of  Marpmann,  Bacillus  sputigqnes  of  Pansini  and  several 
others. 

Type  3. — No  coagulation,  but  formation  of  gas.  The  principal 
representative  of  this  type  is  the  Bacillus  pneumonise  of  Friedlander. 

Type  4- — This  is  represented  by  Bacillus  lactis  inocuus  of  Wilde, 
Bacillus  No.  14  of  Conn,  Bacterium  cocciform  of  Migula,  and  others. 
Neither  coagulation  nor  gas  is  produced. 

BACILLUS  LACTIS  Viscosus. — There  are  several  other  types  of 
organisms  in  the  first  group  of  lactic-acid  producers  which  make  milk 
slimy  or  which  liquefy  the  casein  subsequent  to  its  coagulation.  Many 
bacteria  of  the  first  group  are  so  intimately  related  to  the  colon  bacillus 
that  a  separation  from  it  becomes  impossible.  The  most  important 
organism  of  the  kind  which  make  milk  slimy  or  ropy  is  the  Bacillus 
lactis  viscosus  of  Adametz.  The  organism  has  been  isolated  from 
water  which  probably  is  its  normal  habitat  and  from  which  it  gets 
into  milk.  Ward  has  always  found  this  organism  in  slimy  milk.  It 
grows  at  very  low  temperatures,  better  than  at  higher  temperatures, 
and  for  this  reason  its  multiplication  is  favored  by  the  rapid  cooling 
of  milk.  It  possesses  a  gelatinous,  slimy  capsule  and  forms  zoogleal 
masses;  it  is  this  property  which  imparts  to  milk  the  slimy,  ropy 
character.  Such  milk  is  probably  not  unwholesome,  but  it  is  repulsive 
and  disliked  by  consumers. 

Bacteria  of  Group  II. — The  second  group,  known  as  the  strepto- 
coccus group,  comprises  cocci  which  are  not  motile,  form  no  spores, 
are  either  round,  semiglobular  or  oval  and  form  shorter  or  longer 
chains.  They  are  Gram  positive  and  frequently  show  a  capsule. 
There  are  pathogenic  and  non-pathogenic  bacteria  in  this  group,  the 
former  growing  best  at  37°  C.,  the  latter  at  30°  to  35°  C.  They 
develop  both  aerobically  and  anaerobically.  They  generally  form 
much  lactic  acid  and  coagulate  milk;  sometimes  both  processes  occur 
slowly.  Coagulation  is  sometimes  brought  about  by  an  enzyme  of 
the  rennet  type  without  the  formation  of  acid.  The  lactic  acid 
formed  is  generally  of  the  dextrogyr  variety.  These  bacteria  also 


474  THE  LACTIC  ACID  BACTERIA 

split  up  other  sugars,  but  they  generally  form  lactic  acid  only,  rarely 
other  acids.  This  group  again  contains  a  number  of  types  distin- 
guished and  represented  as  follows : 

Type  1. — Streptococcus  mastiditis  coagulates  milk  and  forms  gas. 

Type  2. — Streptococcus  Guentheri  or  Leichmann  and  Strepto- 
coccus lacticus  Kruse  both  coagulate  milk,  but  do  not  form  gas. 
These  two  organisms  are  probably  the  most  common  and  most  important 
lactic  acid  bacteria  of  milk. 

Type  3. — Streptococcus  Kefir,  does  not  coagulate  milk  and  does 
not  form  gas. 

Type  4- — Streptococcus  lactis  inocuus,  does  not  coagulate  and  does 
not  form  gas. 

Type  5. — Leuconstoc  mesentericus  and  Micrococcus  mucilaginosus 
of  Schiitz  and  Ratz,  which  make  milk  slimy. 

Type  6. — Streptococcus  mirabilis  Roscoe,  which  is  an  arborescent 
organism. 

Type  7. — Streptococcus  coli  gracilis  and  coli  brevis,  which  are 
liquefiers. 

Bacteria  of  Group  III. — The  bacteria  of  the  third  group  vary  much 
in  length.  They  are  most  commonly  slender  rods,  2  to  3  micra  long 
and  0.5  to  0.75  micron  wide.  Some  of  them  form  filaments  or  pseudo- 
filaments  50  or  more  micra  long.  They  are  generally  not  motile, 
never  form  spores,  rarely  show  a  capsule,  and  are  Gram  positive. 
They  are  either  preferably  or  even  strictly  anaerobic.  Their  optimum 
temperature  is  generally  quite  high,  between  40°  and  50°  C.,  their 
minimum  at  25°  C.  or  somewhat  lower.  Milk  is  generally  coagulated 
very  slowly;  the  lactic  acid  formed  is  generally  sinistrogyr.  None 
of  them  are  disease  producers.  They  are  divided  into  types,  the 
most  important  of  which  are : 

Type  1. — Bacillus  casei  of  Freudenberg. 

Type  2. — Bacterium  casei  of  Leichmann. 

Type  3. — Bacterium  caucasicum. 

Type  4.— Bacillus  Delbrucki. 

Type  5. — Bacillus  Aderholdi. 

Type  6. — Bacillus  lactis  acidi  Leichmann. 
There  are  no  liquefying  bacteria  represented  in  this  group. 

Bacteria  of  Group  IV. — The  bacteria  of  the  fourth  group  are  of  the 
type  of  the  Micrococcus  pyogenes  Rosenbach  or  Micrococcus  lactis 
acidi.  They  are  cocci  varying  in  size  from  0.8  to  1.6  micron.  They 
are  single,  in  pairs,  or  in  irregular  groups  (staphylococci).  They  do 
not  form  spores,  are  Gram  positive,  have  their  optimum  of  growth 
between  20°  to  30°  C.,  and  multiply  best  in  the  presence  of  oxygen. 
Some  of  them  liquefy  gelatin,  others  do  not.  Most  of  them  coagulate 
milk.  Formation  of  gas  is  rare.  The  types  in  this  group  are  repre- 
sented by  the  following  bacteria: 

Type  1. — Micrococcus  acidi  Leischmann. 

Type  2. — Micrococcus  lactis  acidi  Marpmann. 


ANAEROBIC  BUTYRIC-ACID  FORMERS  475 

Type  3. — Micrococcus  butyri  aromafaciens. 

Type  4- — Micrococcus  candicans  Fliigge. 

Type  5. — Micrococcus  lactis  viscosi  Gruber  (causing  slimy  or  ropy 
milk). 

Type  6. — Micrococcus  coronatus  Fliigge. 

Type  7. — Micrococcus  cirrhiformis  Migula  (forms  gas). 

The  Coli  Aerogenes  Bacteria. — Under  the  preceding  four  collective 
groups  of  lactic-acid  bacteria  proper  a  number  of  microorganisms 
have  been  mentioned.  Those  named,  however,  are  only  a  small 
fraction  of  the  bacteria  of  the  groups  which  now  comprise  several 
hundred,  though  it  is  very  probable  that  many  which  in  fact  are 
identical  species  have  been  described  by  different  observers  under 
different  names.  Besides  these  lactic-acid  bacteria  par  excellence, 
others,  such  as  the  Bacillus  coli  communis  and  the  Bacillus  lactis 
aerogenes  of  Escherich,  form  lactic  acid  from  sugar  of  milk.  The 
Bacillus  coli  communis  has  been  fully  described  in  a  previous  chapter 
as  evidently  including  a  number  of  varieties. 

The  Bacillus  aerogenes  or  the  Bacterium  lactis  aerogenes  of 
Escherich  is  generally  plumper  and  shorter  than  the  Bacillus  coli 
communis.  It  is  1  to  2  micra  long  and  0.5  to  1  micron  wide;  it 
presents  itself  singly,  in  pairs,  rarely  in  chains,  or  pseudofilaments. 
It  is  not  motile,  does  not  form  spores,  and  possesses  no  flagella.  On 
gelatin  it  forms  large,  white,  not  transparent  colonies.  It  splits 
glucose  with  the  formation  of  gas.  It  also  ferments  lactose  in  milk, 
but  forms  from  it  more  acetic  than  lactic  acid.  It  also  forms  succinic 
acid.  Such  lactic-acid  bacteria  proper  as  the  Streptococcus  Guentheri 
and  Streptococcus  lacticus  in  their  growth  in  milk  largely  prevent  the 
development  of  the  bacteria  of  the  coli  aerogenes  group. 


ANAEROBIC  BUTYRIC-ACID  FORMERS. 

Anaerobic  bacteria  which,  in  addition  to  lactic  acid,  also  form 
butyric  acid  from  lactose  are  frequently  found  in  milk.  The  first 
bacterium  of  this  group  was  discovered  by  Pasteur,  who  also  recog- 
nized its  anaerobic  nature.  Prazmowski  first  introduced  the  name  of 
Clostridium  butyricum  for  a  bacterium  of  this  group ;  later  a  number, 
several  of  which  were  evidently  identical,  were  described  under  various 
names.  Beyerinck  united  the  butyric-acid  bacteria  into  a  family 
under  the  name  of  granulobacter,  or  amylobacter,  because  these 
organisms  when  growing  in  a  medium  containing  starch  form  char- 
acteristic granules  in  their  interior.  A  number  of  very  important 
anaerobic  pathogenic  bacteria  belong  to  this  family,  such  as  the 
bacillus  of  black  leg,  the  bacillus  of  malignant  edema,  and  others 
described  in  previous  chapters. 

Schattenfroh  and  Grassberger  have  classified  the  butyric-acid 
formers  into  four  groups,  as  follows : 


476  ANAEROBIC  BUTYRIC-ACID  FORMERS 

1.  Motile  butyric-acid  bacteria. 

2.  Gas  formers  of  the  black-leg  bacillus  type,     (a)  Spore-forming 
(black-leg  bacillus,  Bacillus  aerogenes  capsulatus);    (6)   non-motile 
(non-pathogenic)  butyric-acid  bacilli. 

3.  Organisms  of  the  type  of  the  malignant  edema  bacillus. 

4.  Putrefying  butyric-acid  bacilli  of  the  type  of  Bacillus  putrificus 
Bienenstock. 

The  motile  butyric-acid  bacillus  is  relatively  prevalent  as  a  sapro- 
phyte in  soil,  water,  grain,  flour,  cheese,  more  rarely  in  milk,  which 
generally  does  not  form  a  favorite  soil  for  its  development.  It  grows 
best,  according  to  Beyerinck,  in  artificial  culture  in.  a  5  per  cent. 
peptone  solution  (under  anaerobic  conditions).  It  is  a  long,  slender 
rod,  motile,  and  with  flagella  surrounding  the  entire  body.  Before 
sporulation  the  bacillus  forms  granulose1  from  starch  in  its  interior 
and  assumes  the  clostridium  shape.  The  spore  escapes  from  its 
membrane  generally  at  one  end,  and  the  empty  shell  may  for  some 
time  remain  over  one  end  of  the  young  bacillus  like  a  cap.  The 
spores  are  killed  when  exposed  in  boiling  water  for  three  minutes  to 
100°  C.  On  gelatin  the  organism  forms  cloudy,  hazy  colonies;  the 
colonies  may  also  be  better  defined  and  surrounded  by  filamentous 
excrescences  or  they  may  form  a  veil  on  the  surface  of  the  medium 
without  any  distinctly  defined  boundaries  whatever.  The  bacillus, 
while  fermenting  dextrose,  saccharose,  lactose,  starch,  and  glycerin, 
and  forming  from  them  butyric  acid,  lactic  acid,  carbon  dioxide,  and 
hydrogen,  does  not  split  up  albumin.  Butyric  acid  is  formed  in 
excess  of  lactic  acid  and  hydrogen  in  excess  of  carbon  dioxide.  In 
milk  a  floating  layer  of  casein  full  of  gas-bubbles  is  formed.  This 
motile  butyric-acid  bacillus  is  identical  with  the  bacilli  described 
under  the  following  names:  Bacillus  amylobacter  I  and  II,  Gruber, 
Granulobacter  saccharobutyricus  Beyerinck,  and  Bacillus  saccharo- 
butyricus  of  Klecki. 

The  non-motile  butyric-acid  bacillus  is  likewise  frequently  found 
in  nature,  and  as  it  is  a  regular  inhabitant  of  the  feces  of  cattle,  often 
in  milk.  According  to  Botkin-Rodella  it  can  be  easily  obtained  by 
inoculating  sterile  milk  covered  by  a  cream  layer  10  cm.  high  from 
garden  earth.  The  milk,  while  still  heated  to  70°  C.,  is  inoculated 
and  then  kept  in  the  incubator,  when  the  non-motile  bacillus  generally 
grows  abundantly.  The  organism  occurs  in  two  types.  The  first 
type  presents  cylindrical  rods,  with  rounded  ends,  generally  forming 
chains  of  three  to  six  links,  or  pseudofilaments  20  to  50  micra  long. 
Its  colonies  are  small,  very  shining,  and  surrounded  by  numerous 
excrescences.  The  second  type  forms  shorter  and  more  slender  rods, 
rarely  arranged  in  longer  chains,  and  its  colonies  are  round,  sharply 
defined,  smooth,  and  dewdrop  like.  Both  types  in  starch-agar  form 


1  Granulose  formed  from  starch  in  the  interior  of  the  bacilli  of  this  group  like  starch  stains 
blue  with  iodin  solution. 


PEPTONIZING  BACTERIA  IN  MILK  477 

granulose  in  their  interior  and  spores.  The  latter  can  resist  boiling 
in  water  at  100°  C.  for  one  and  one-half  hours.  These  organisms 
liquefy  gelatin  and  coagulate  milk.  Grassberger  claims  that  the  non- 
motile  butyric  acid  bacilli  are  black-leg  bacilli  without  pathogenic 
properties. 

The  Bacillus  putrificus  Bienenstock  is  a  slender  rod,  5  to  6  micra 
long,  with  rounded  ends.  In  gelatin,  which  is  liquefied,  it  forms  long 
chains  and  pseudofilaments.  The  spores  are  formed  at  one  end  like 
those  of  the  tetanus  bacillus  and  the  sporulating  organism  has  the 
drum-stick  appearance.  This  is  best  shown  on  agar  or  blood-serum 
cultures.  The  spores  can  withstand  heating  at  80°  C.  for  three 
minutes,  but  they  are  killed  in  boiling  water  in  five  minutes.  On 
agar  slants  kept  under  anaerobic  conditions  the  organism  forms  a 
transparent  veil  and  clouds  the  mass  of  the  medium,  also  the  water 
of  condensation.  The  organism  may  be  obtained  from  hard  cheese, 
ground  up  fine  and  incubated  with  a  nutrient  bouillon,  containing 
0.5  c.c.  lactic  acid,  kept  under  anaerobic  conditions  in  the  incubator 
for  ten  to  fourteen  days. 

Other  butyric-acid  bacteria  which  have  frequently  been  found  in 
milk  are  the  Bacillus  lactopropylbutyricus  of  Tissier  and  Gashing, 
the  Clostridium  Pastorianum  Winogradsky,  and  the  Clostridium 
Americanum  of  Pringsheim.  The  latter  is  not  as  strictly  anaerobic 
as  the  other  species  enumerated.  This  organism  was  found  on 
American  potatoes. 

A  bacterium  frequently  found  in  soft,  strong-smelling  cheese  is 
the  Paraplectrum  fetidum  of  Weigmann.  It  is  a  rod  from  2.5  to  8 
micra  long,  0.6  micron  thick;  it  forms  spores  in  milk  in  two  to  three 
days,  and  coagulates  this  fluid  in  forty-eight  hours.  The  coagulation 
is  soon  followed  by  a  peptonizing  liquefaction,  with  the  formation 
of  a  very  fetid  smell  similar  to  that  of  limburger  cheese. 

PEPTONIZING  BACTERIA  IN  MILK. 

There  are  two  groups  of  spore-forming,  aerobic  bacteria  in  par- 
ticular which  are  very  widespread  in  nature.  They  secrete  enzymes 
and  almost  invariably  get  into  milk,  in  which  they  coagulate  the  casein 
and  subsequently  liquefy  it  again.  The  coagulation,  brought  about 
by  a  rennet  enzyme,  may  be  only  very  slightly  marked,  because  the 
peptonizing  ferment  is  furnished  by  these  bacteria  so  promptly  and 
evidently  so  abundantly  that  the  peptonizing  and  liquefying  action 
greatly  overshadows  the  coagulation.  The  bacteria  of  this  type  are 
characterized  by  two  representative  species,  the  Bacillus  subtilis,  or 
hay  bacillus,  and  the  Bacillus  mesentericus  vulgatus,  or  common 
potato  bacillus.  They  are  present  in  the  soil,  in  manure,  on  grains, 
potatoes,  hay,  and  straw,  and  in  air  and  water.  They  are  putrefying 
organisms  which,  in  connection  with  anaerobes  with  whom  they  live 
in  symbiotic  community,  are  engaged  in  breaking  up  organic  waste 


478  PEPTONIZING  BACTERIA  IN  MILK 

material  into  very  simple  chemical  compounds.  They  get  into  milk 
during  milking,  with  the  dust  from  hay  and  straw,  and  they  are 
undoubtedly  also  found  on  the  skin  of  the  cow's  udder.  They  are 
not  numerous  in  cow's  feces,  hence  they  do  not  indicate  contamination 
by  manure. 

Bacillus  Subtilis. — The  Bacillus  subtilis  can  be  easily  obtained  by 
making  an  infusion  of  hay,  boiling  it  for  one  hour,  and  incubating  it 
at  37°  C.  The  boiling  destroys  the  vegetative  forms  of  all  kinds  of 
bacteria,  but  does  not  affect  the  spores  of  the  Bacillus  subtilis.  The 
latter  is  a  rod  4  to  5  micra  long  and  0.8  to  1.2  micron  thick;  some- 
times very  short  rods  are  seen,  also  long  chains  and  pseudofilaments. 
The  bacillus  is  motile  and  possesses  eight  to  twelve  flagella,  arranged 
around  the  body  of  the  organism.  The  spores  are  formed  in  the  centre, 
sometimes  more  toward  one  end  of  the  bacillus.  When  germinating 
they  rupture  the  spore  membrane  in  the  equatorial  plane,  not  at 
either  end.  The  elongating  young  bacillus  sometimes  carries  the 
empty  spore  membrane  along,  attached  to  it  like  a  cap.  On  gelatin 
small,  white,  punctate  colonies  are  formed,  which  later  become  darker, 
granular  and  brownish,  and  send  out  hair-like  processes  into  the 
culture  soil.  The  colonies  rapidly  liquefy  gelatin.  On  gelatin  stick 
cultures  rapid  liquefaction  occurs  along  the  whole  line  of  the  stick. 
On  agar  slants  a  heavy,  corrugated  growth  is  formed.  Blood  serum 
is  likewise  liquefied.  The  bacillus  grows  well  and  abundantly  on 
potatoes.  As  already  stated,  it  peptonizes  milk  very  rapidly,  but  its 
growth  in  milk  is  prevented  as  soon  as  lactic-acid  bacteria  have 
formed  a  small  amount  of  lactic  acid.  According  to  Lafar  the  presence 
of  0.1  per  cent,  of  lactic  acid  prevents  the  development  of  the  Bacillus 
subtilis.  It  is  a  strictly  aerobic  organism,  and  requires  for  its  best 
growth  an  abundant  supply  of  oxygen.  The  organism  is  Gram 
positive. 

Bacillus  Mesentericus  Vulgatus. — The  Bacillus  mesentericus  vulgatus 
is  a  widely  prevalent  saprophyte.  It  is  the  most  common  of  the  group 
of  potato  bacilli  and  is  somewhat  smaller  than  the  Bacillus  subtilis. 
It  is  motile  and  possesses  numerous  flagella,  which  generally  are  found 
on  one  side  of  the  body  only.  The  spores  are  elliptical  and  large. 
On  gelatin  the  growth  closely  resembles  that  of  the  Bacillus  subtilis 
and  liquefies  it  energetically.  In  milk  coagulation  is  more  marked 
than  in  the  case  of  the  subtilis,  but  subsequent  liquefaction  is  also 
soon  accomplished.  On  potatoes  the  growth  is  very  abundant  and 
forms  a  thick,  corrugated  layer.  The  organism  occurs  in  several 
varieties,  such  as  the  Bacillus  mesentericus  fuscus,  which  forms  a 
grayish-brown  pigment,  and  the  Bacillus  mesentericus  ruber,  which 
forms  a  reddish-yellow  pigment.  The  Bacillus  liodermes,  or  rubber 
bacillus  of  Loeffler,  which  also  belongs  to  the  same  group,  forms 
a  rubber-like  growth  on  potatoes. 

The  common  root  bacillus,  Bacillus  mycoides,  so  called  on  account 
of  its  rizoid  cultures  on  gelatin,  is  also  frequently  found  in  milk. 


ALCOHOLIC  FERMENTATION  OF  MILK  479 

Other  Peptonizing  Bacteria. — A  number  of  other  organisms  fre- 
quently found  in  milk,  which  coagulate  the  casein  and  subsequently 
peptonize  or  liquefy  it,  have  been  described  by  Duclaux.  These 
are  the  Tyrothrix  or  Bacillus  tenuis,  Tyrothrix  or  Bacillus  distortus, 
Tyrothrix  or  Bacillus  geniculatus  and  several  others  of  the  same  family. 
These  organisms  are  aerobic  and  facultative  anaerobic,  spore  forming, 
motile  or  immobile  rods.  Tyrothrix  geniculatus  peptonizes  the  casein 
without  preceding  precipitation. 

It  is  generally  believed  that  the  bacteria  of  the  Bacillus  subtilis 
group  play  an  important  role  in  the  ripening  of  cheese;  also  the 
different  species  of  tyrothrix  described  by  Duclaux. 


CHROMOGENIC  BACTERIA  IN  MILK. 

Fluorescent  and  chromogenic  bacteria  sometimes  impart  a  par- 
ticular color  to  milk.  The  former  kind  sometimes  produce  a  green 
fluorescent  pigment.  The  Bacillus  cyanogenus,  a  motile,  Gram- 
negative,  non-sporogenous  bacterium,  produces  a  bluish  to  brownish- 
red  pigment.  The  Bacillus  violaceus,  a  water  bacterium,  sometimes 
gets  into  milk  and  produces  a  violet  color,  as  does  also  the  Bacillus 
membranaceus  amethystinus.  Sarcina  rosacea  stains  cream  pinkish, 
while  the  Bacillus  prodigiosus  may  produce  in  cream  a  more  decidedly 
red  color.  Bacillus  lactorubefaciens  stains  the  whole  milk  red  and 
makes  it  slimy,  Bacillus  mycoides  roseus  gives  it  a  rust-brown  color, 
Bacterium  synxanthum  stains  it  yellow. 


ALCOHOLIC  FERMENTATION  OF  MILK. 

Under  certain  conditions  yeast  cells,  or  saccharomyces,  are  found 
in  milk.  They  possess  the  faculty  of  forming  alcohol  from  lactose. 
The  enzyme  which  brings  about  this  fermentation  is  called  lactose, 
and  is  not  identical  with  the  yeast  enzyme  known  as  zymase,  which 
splits  monosaccharids  into  alcohol  and  carbon  dioxide.  Most  of  the 
microorganisms  which  form  alcohol  from  lactose  are  not  true  yeast 
cells,  or  saccharomyces,  but  belong  to  the  nearly  related  family  known 
as  Torula.  The  principal  generic  difference  between  yeast  cells  and 
torula  is  that  the  former  form  spores,  but  the  latter  are  asporogenous , 
The  most  important  organisms  which  form  alcohol  in  milk  from  milk 
sugar  are  the  following: 

Saccharomyces  lactis  acidi  of  Grotenfeld  acidulates  milk,  coagulates 
it,  and  forms  some  alcohol;  Saccharomyces  Freudenreich  and  Jensen, 
Saccharomyces  fragilis  of  Joergensen,  and  Torula  lactis  of  Adametz. 
The  latter  is  a  torula  which  was  discovered  by  Weigmann.  It  forms 
in  milk  from  lactose  51.2  per  cent,  by  weight  of  alcohol,  34.4  per 
cent,  of  carbon  dioxide  and  3.6  per  cent.of  butyric  acid.  The  so-called 


480  ALCOHOLIC  FERMENTATION  OF  MILK 

kefir  or  kafyr  granules,  which  are  used  in  the  Orient  to  prepare  an 
alcoholic  beverage  from  milk,  contain  both  bacteria  and  yeast  cells. 
The  cause  of  the  fermentation  of  the  alcoholic  beverage,  known  as 
kumys,  prepared  in  Siberia  from  mare's  milk  and  ass's  milk,  is  not 
known. 

In  addition  to  the  bacteria,  saccharomyces  and  torula,  higher 
moulds  (i.  e.,  those  forming  a  true  mycelium)  are  likewise  found  in 
milk.  Among  such  moulds  may  be  mentioned  Oidium  lactis,  Peni- 
cillium  glaucum,  Penicillium  roqueforti,  Penicillium  camemberti,  and 
Mucor  racemosus.  Many  of  these  moulds  play  an  important  role 
in  the  ripening  and  flavoring  of  cheese. 


QUESTIONS. 

1.  Is  it  possible  in  practice  to  obtain  germ-free  milk  from  the  cow  in  larger 
amounts?    If  not,  why  not? 

2.  What  measures  are  necessary  to  keep  the  bacterial  content  of  milk  as  low 
as  possible? 

3.  What  is  the  most  important  source  of  increase  of  the  bacterial  contents 
of  milk.  ? 

4.  What  should  be  the  maximum  of  dirt  permissible  in  milk? 

5.  What  brings  about  the  souring  of  milk?    What  change  occurs  in  lactose 
or  sugar  of  milk  in  the  process  of  souring? 

6.  What  is  meant  by  a  stereo-isomenc  body? 

7.  What  is  meant  by  dextrogyr  lactic  acid?    What  is  meant  by  sinistrogyr 
lactic  acid? 

8.  What  is  polarized  light? 

9.  Where  are  the  lactic  acid  bacteria  found? 

10.  Describe  in  general  terms  the  four  groups  of  lactic-acid  bacteria. 

11.  Name  some  of  the  various  types  belonging  to  each  of  the  four  groups. 

12.  What  are  the  morphologic  features  of  the  most  common  types  of  lactic 
acid  bacteria? 

13.  What  is  meant  by  the  coli-aerogenes  bacteria? 

14.  What  are  the  anaerobic  bacteria  found  in  milk? 

15.  What  are  the  four  groups  of  butyric  acid  formers? 

16.  What  is  a  clostridium? 

17.  Describe  some  of  the  butyric-acid  formers. 

18.  What  peptonizing  bacteria  commonly  occur  in  milk? 

19.  Describe  the  Bacillus  subtilis. 

20.  Describe  the  Bacillus  mesentericus  vulgatus. 

21.  Name  and  describe  some  of  the  chromogenic  bacteria  found  in  milk. 

22.  Name  some  organisms  producing  alcohol  in  milk. 


CHAPTEE    XLVI. 

THE  BACTERIOLOGY  AND  THE  BACTERIOLOGIC  EXAMINATION  OF 
MILK    (CONTINUED)— PATHOGENIC    BACTERIA    IN    MILK— THE 
TUBERCLE  BACILLUS— METHODS  FOR  DETERMINING  ITS 
PRESENCE  IN  MILK— HUMAN  AND  OTHER  CATTLE 
DISEASES  TRANSMISSIBLE  THROUGH  MILK- 
NUMBER  AND    SIGNIFICANCE    OF 
LEUKOCYTES  IN  MILK. 

PATHOGENIC  BACTERIA  IN  MILK. 

THE  organisms,  such  as  the  lactic-acid  bacteria,  the  butyric-acid 
bacteria,  the  alcohol  formers,  and  the  higher  moulds,  which  have 
already  been  discussed,  are  saprophytes  and  not  disease  producers. 
Pathogenic  bacteria,  however,  also  occur  in  milk.  They  may  be 
derived  directly  from  the  cow,  from  those  handling  the  milk,  or 
they  may  be  accidentally  introduced  with  water  or  otherwise. 

Tubercle  Bacillus. — Of  the  microorganisms  pathogenic  to  man 
which  may  occur  in  milk,  tubercle  bacilli  are  the  most  important. 
It  is  possible  to  keep  disease-producing  bacteria,  such  as  the  typhoid, 
diphtheria,  and  other  bacilli  out  of  milk  by  having  the  proper  persons 
collect  it  properly  in  sterile  vessels;  but  tubercle  bacilli  which  come 
from  the  cow  cannot,  under  certain  conditions,  be  kept  out  of  the  milk. 
This  is  undoubtedly  the  case  in  tuberculosis  of  the  udder  itself  and  in 
advanced  general  tuberculosis.  Examinations  of  market  milk  for  the 
presence  of  tubercle  bacilli  have  been  made  in  many  cities  and  the 
positive  findings  vary  greatly.  Anderson,  in  223  specimens  of  milk 
examined  in  Washington,  D.  C.,  found  15  (6.27  per  cent.)  with  live, 
virulent,  tubercle  bacilli;  Hess,  in  New  York,  in  106  samples  found  17 
(16  to  17  per  cent.);  Delepine,  in  Manchester,  in  125  samples  found 
22  (17.6  per  cent.);  Eber,  in  Leipzig,  in  210  samples  found  22 
(10.5  per  cent.);  Klein,  in  London,  in  100  samples  found  7;  and  Petri, 
in  Berlin,  in  86  samples  found  33  (38.4  per  cent.).  Very  low  figures  are 
reported  from  Italy  where  in  a  number  of  cities  no  tubercle  bacilli 
were  found  in  any  of  the  samples  examined;  also  from  Wiirtemberg, 
where  Herbert  examined  101  samples  without  any  positive  findings. 
The  highest  findings  have  been  reported  by  Kanthack  and  Sladen,  who 
found  9  samples  out  of  16  infected  in  England,  and  by  Obermiiller, 
in  Berlin,  who  took  14  samples  from  one -place  and  found  them  all 
infected.  Schroeder  states  that  most  tubercle  bacilli  get  into  milk 
from  fecal  contamination.  He  reports  that  he  has  been  able  to  show 
in  a  number  of  experiments  their  presence  in  the  feces  of  cows  which 
31 


482  PATHOGENIC  BACTERIA  IN  MILK 

were  in  good  physical  condition  and  in  which  the  diagnosis  of  tuber- 
culosis could  be  made  only  by  the  tuberculin  test.  He  also  calls 
attention  to  the  fact  that  tubercle  bacilli  may  not  be  voided  contin- 
ually by  such  animals,  and  that  they  may  occur  in  the  milk  from 
certain  dairies  and  sources  only  on  a  few  days  during  several  weeks. 
Eber  has  made  similar  observations  in  regard  to  the  intermittent 
distribution  of  tubercular  milk  by  dairies  in  Leipzig.  Schroeder  seems 
to  believe  that  tubercle  bacilli  may  be  excreted  with  the  feces  of 
tubercular  cows  and  otherwise,  even  in  the  absence  of  open  tubercular 
lesions,  but  this  view  is  certainly  not  shared  by  many.  At  the  Eighth 
International  Congress  in  Washington,  Bang  stated  that  when  the 
tubercular  lesions  in  a  cow  are  closed  no  tubercle  bacilli  are  present 
in  either  the  milk  or  the  feces.  When  some  of  the  lesions  are  open 
and  the  bacilli  enter  into  the  lymph  or  blood  circulation  the  bacilli 
may  be  temporarily  present  in  the  milk  or  feces.  Heymans  believes 
that  Schroeder's  results  with  the  inoculations  of  feces  into  guinea-pigs 
are  not  quite  conclusive  as  to  the  fecal  excretion  of  tubercle  bacilli  in 
an  early  stage  of  tuberculosis  in  cattle. 

In  considering  the  significance  of  bovine  tubercle  bacilli  in  milk 
it  must  be  borne  in  mind  that  the  presence  of  a  small  or  even  a  larger 
number  of  bacilli  sufficient  to  produce  tuberculosis  in  a  guinea-pig 
by  intraperitoneal  or  subcutaneous  injection1  may,  but  generally  will 
not,  produce  tuberculosis  in  man,  even  when  ingested  as  food  for  a 
considerable  period.  That  this  is  indeed  the  case  has  been  repeatedly 
shown  by  a  number  of  observers,  and  the  following  investigations  are 
of  interest: 

Hess,  of  the  Research  Laboratory  of  the  Health  Department  of 
New  York  City,  obtained  specimens  of  raw  milk  in  New  York  from 
large  forty-quart  cans.  He  collected  this  milk  from  dealers  who  had 
small  children  who  drank  the  milk  in  a  raw  state.  He  secured  107 
specimens,  and  in  17  of  these  tubercle  bacilli  able  to  kill  guinea-pigs 
were  found;  8  stems  were  subsequently  obtained  from  the  dead 
animals  and  7  were  found  to  be  of  the  bovine  and  1  of  the  human  type. 
This  induced  the  author  to  point  out  that  tubercle  bacilli  in  milk 
might  occasionally  be  derived  from  man  and  not  from  cattle.  Of  the 
dealers  who  had  dispensed  the  milk  containing  tubercle  bacilli,  ten 
were  found  to  have  18  children  who  had  been  regularly  fed  on 
this  raw  milk;  9  of  the  children  were  two  years  or  under  and  only 
1  was  over  five  years.  The  children  were  carefully  watched  for 
one  year,  and  16  were  submitted  to  the  Pirquet  tuberculin  test; 
4  reacted  in  a  positive  manner;  of  the  4,  2  were  perfectly  healthy 
and  2  were  poorly  nourished,  but  showed  no  definite  signs  of  tuber- 
culosis. Of  the  2  in  poor  condition,  1  had  five  months  previously  had 

1  Knutt  has  shown  that  a  tubercle-bacilli-containing-milk  which  would  produce  tuberculosis 
in  guinea-pigs  in  intraperitoneal  inoculations  of  0.00001  gram  (i.  e.,  a  dose  of  T^  of  a  milligram) 
would  only  produce  tuberculosis  if  fed  in  a  dose  of  15  grams,  i.  e. ,  one  million  and  a  half  (1 ,500,000) 
times  the  intraperitoneal  dose. 


LIVE  TUBERCLE  BACILLI  IN  MILK  483 

a  cervical  adenitis.1  From  the  fact  that  almost  all  of  the  children 
who  had  been  regularly  drinking  infected  milk  were  found  in  good 
health,  the  author  concluded  that  milk  containing  bovine  tubercle 
bacilli  does  not  necessarily  or  even  usually  excite  tuberculosis  in  children. 
On  account  of  the  extent  and  importance  of  the  discussion  of  the 
transmissibility  of  bovine  tuberculosis  to  man,  the  German  Imperial 
Health  Office  instituted  a  collective  investigation  of  a  number  of 
cases  in  which  milk  from  cows  suffering  with  tuberculosis  of  the 
udder  was  regularly  and  for  a  long  period  of  time  consumed  raw. 
Kossel  has  recently  published  a  summary  of  the  results  of  the  inves- 
tigation. It  was  found  that  360  persons  had  consumed  such  milk; 
of  these  200  were  adults,  151  children,  age  not  given  for  9.  Two 
children  aged  one  year  and  ten  months,  and  one  year  and  three 
months,  respectively,  showed  undoubted  evidences  of  infection  with 
bovine  tuberculosis. 

In  both  the  cervical  glands  were  affected,  and  in  the  younger  child 
tubercle  bacilli  of  the  bovine  type  could  be  demonstrated.  In  examin- 
ations, however,  respectively  one  and  one-half  and  two  and  one-half 
years  subsequent  to  the  first  examination  both  children  showed  a 
healthy  development  and  a  good  appearance,  notwithstanding  the 
fact  that  they  had  been  fed  on  raw  milk  from  cows  with  advanced 
udder  tuberculosis.  Four  adults  and  8  other  children  which  had 
habitually  consumed  the  same  raw  milk  remained  well.  In  12  cases 
of  the  360,  tuberculous  infection  was  suspected,  but  examination  made 
months  and  years  after  the  first  did  not  reveal  any  evidence  of  gland- 
ular tuberculosis;  in  fact,  the  gland  swellings  had  disappeared  and 
some  of  the  physicians  who  had  made  the  earlier  examinations  doubted 
the  correctness  of  their  former  diagnosis.  In  reviewing  the  results  of 
this  investigation,  Weber  concludes  that  the  danger  to  man  from  raw 
milk  from  tubercular  cows  is  very  small  in  comparison  with  the  danger 
from  persons  with  open  pulmonary  tuberculosis,  and  he  agrees  with 
Fliigge  and  Osterman's  claim  that  great  numbers  of  tubercle  bacilli 
must  be  present  before  much  danger  of  infection  exists. 

Methods  for  the  Determination  of  the  Presence  of  Live  Tubercle  Bacilli 
in  Milk. — As  milk  and  milk  products,  such  as  cream  and  butter,  fre- 
quently contain  acid-fast  saprophytes  which  cannot  morphologically 
be  distinguished  from  the  tubercle  bacillus,  the  usual  staining  method 
used  for  exhibiting  this  organism  can  only  be  considered  a  preliminary 
procedure,  and  the  finding  of  acid-fast  bacilli  must  be  followed  by  a 
proper  inoculation  of  the  suspected  material.  The  steps  in  the 
complete  examination  are  the  following : 

I.  Centrifuge  the  milk  in  an  electric  centrifuge  for  five  to  ten 
minutes.  A  layer  of  cream  is  then  formed  on  top  and  a  sediment  at 
the  bottom  of  the  tube. 

1  It  was  ascertained  in  a  subsequent  examination  that  the  tubercle  bacilli  responsible  for 
the  cervical  adenitis  in  this  child  were  of  the  typus  humanus.  (Personal  communication  from 
Doctor  W.  H.  Park,  Director  of  the  New  York  Health  Research  Laboratory.) 


484  PATHOGENIC  BACTERIA  IN  MILK 

II.  Remove  carefully  with  a  small  sterile  spoon  (it  is  best  to  use  a 
platinum  spoon)  the  fat  from  the  top  and  place  it  in  a  sterile  covered 
watch-glass  or  Petri  dish. 

III.  Pour  off  the  fluid  layer  forming  the  middle  stratum,  then  like- 
wise, preserve  the  sediment  in  a  sterile  vessel. 

IV.  The  cream  and  the  sediment  may  be  mixed  or  examined 
separately.     In  very  exact  examinations  it  is  best  to  inoculate  a 
mixture  of  cream  and  sediment  of  each  sample  of  milk  into  a  guinea- 
pig  or  several  of  these  animals.    This  is  rarely  done,  as  it  requires 
too  many  experimental  animals  and  too  much  really  unnecessary 
labor. 

As  a  rule,  several  cover-glasses  are  prepared  from  the  mixed  creams 
and  sediments,  and  they  are  examined  by  the  Ziehl-Neelson  method 
of  staining  with  carbol-fuchsin  solution.  The  samples  which  contain 
acid-fast  bacilli  are  diluted  with  sterile  physiologic  salt  solution  and 
inoculated  subcutaneously  into  guinea-pigs,  best  in  the  neighborhood 
of  some  lymph  gland  (inguinal  glands)  with  the  suspected  material. 
In  experiments  for  the  detection  of  live  tubercle  bacilli  in  milk, 
butter,  etc.,  the  inoculations  should  always  be  made  subcutaneously, 
not  intrapejritoneally,  because  in  the  latter  case  guinea-pigs  generally 
develop  pseudotuberculous  lesions  in  the  presence  of  the  acid-fast 
bacilli  of  Moller,  Rabinowitsch  and  others,  even  when  tubercle 
bacilli  are  absent. 

For  the  microscopic  examination  of  butter  for  acid-fast  bacilli 
the  following  procedure  is  recommended  by  Roth : 

I.  Two  to  4  grams  of  butter  are  placed  in  a  sterile  centrifuge  tube, 
which  is  filled  about  three-quarters  full  with  sterile,  distilled  water. 

II.  This  tube  so  prepared  is  placed  in  a  water  bath  and  kept  at 
50°  C.  until  all  of  the  butter  is  melted. 

III.  The  tube  is  now  closed  with  a  cork  or  glass  stopper  and  well 
shaken  for  some  time  to  mix  the  fat  and  water  thoroughly.    It  is  now 
placed  into  the  incubator  in  an  inverted  position,  i.  e.,  with  the  cork 
or  glass  stopper  down  and  the  pointed  end  of  the  centrifuge  tube  up. 
After  all  the  fat  has  risen  to  what  is  now  the  upper  end,  the  tube 
is  left  in  a  cool  place  until  the  butter  fat  has  again  solidified. 

IV.  The  tube  is  now  inverted,  the  cork  or  stopper  opened,  the 
wash  water  which  now  contains  most  of  the  bacteria  is  poured  into 
another  sterile  centrifuge  tube,  and  this  is  again  centrifuged. 

V.  The   sediment  is  then  used  for  cover-glass  preparations,  and 
these,  when  air  dry,  are  washed  for  a  short  time  in  an  absolute 
alcohol-ether  mixture. 

VI.  The  cover-glass  preparations  are  then  stained  and  decolorized 
in  the  usual  manner. 

Typhoid  Bacillus. — The  bacilli  which  cause  the  human  disease 
typhoid  fever  are  voided  in  enormous  numbers  during  and  after  the 
course  of  the  affection  with  the  feces.  By  getting  into  wells  supplying 
farms  and  dairies  from  vaults,  cesspools,  and  other  sources,  or  by 


TYPHOID  BACILLUS  485 

contaminating  ponds,  brooks,  and  rivers  they  may  pollute  water,  and 
by  this  means  the  disease  is  generally  spread  to  human  beings.  It  has 
also  been  ascertained  that  certain  persons,  after  having  passed  through 
an  attack  of  typhoid  fever  may  harbor  numerous  typhoid  bacilli  in 
their  intestines  for  years  without  injury  to  themselves  and  discharge 
them  in  their  stools.  Such  persons  who  have  been  called  "permanent 
carriers"  may  become  a  source  of  great  danger  to  their  surroundings 
or  to  the  water  supply  of  a  larger  territory. 

Most  typhoid  epidemics  are  waterborne,  but  epidemiological 
statistics  have  also  shown  that  a  high  percentage  of  typhoid  epidemics, 
preferably  those  in  a  limited  territory,  are  milkborne.  Typhoid 
bacilli  generally  find  their  way  into  milk  through  contaminated  water. 
Such  water  being  used  cold  to  wash  out  milk  cans  and  a  sufficient 
quantity  may  remain  in  the  container  to  bring  about  infection,  as  milk 
is  a  favorable  culture  medium  for  typhoid  bacilli.  When  typhoid 
bacilli  get  into  raw  milk  there  is  first  a  marked  decrease  in  their 
number,  but  later  a  great  increase  which,  however,  does  not  lead  to 
such  changes  as  acid  formation  and  coagulation,  which  might  betray 
its  abnormal  character.  If,  however,  the  lactic-acid  bacteria  have 
greatly  increased  in  number  and  have  produced  a  marked  acidity 
typhoid  bacilli  are  first  inhibited  in  their  growth  and  later  killed  off 
entirely.  Bassenge  states  that  a  degree  of  acidity  of  0.3  to  0.4  per  cent, 
in  milk  will  kill  typhoid  bacilli  after  twenty-four  hours.  They  survive, 
however,  for  a  long  time  in  fresh,  raw  milk  which  has  been  infected 
and  is  kept  at  a  low  temperature.  The  bacilli  perish  rapidly  in  sour 
cream,  but  they  may  survive  for  a  considerable  time  in  sweet  cream 
and  in  butter.  Schiider,  who  investigated  638  smaller  and  larger 
epidemics  of  typhoid  fever  and  12  individual  cases,  came  to  the 
conclusion  that  the  disease  was  due  to  water  462  times  (70.8  per  cent.) 
and  to  milk  111  times  (17.0  per  cent.).  Trask,  in  analyzing  179 
typhoid  epidemics,  chiefly  in  the  United  States  and  England,  as  spread 
by  milk,  found  that  113  were  traceable  to  farms,  dairies,  or  milk  shops. 
These  conclusions,  however,  rest  solely  upon  epidemiological  data, 
because  typhoid  bacilli  have  only  very  rarely  been  found  in  milk. 
Vaughan  reported  the  finding  of  typhoid  bacilli  in  milk  in  1890,  but 
at  that  time  the  bacilli  were  not  indubitably  identified;  in  1906, 
however,  Konradi  isolated  bacilli  from  milk  which  were  fully  identified 
by  agglutination  and  other  tests;  Shoemaker  also  obtained  typhoid 
bacilli  from  milk,  handled  by  a  person  convalescent  from  typhoid 
fever.  Flies  have  undoubtedly  conveyed  typhoid  bacilli  from  feces  to 
milk.  Levy  and  Jacobstal  claim  to  have  isolated  typhoid  bacilli  from 
a  large  abscess  in  the  spleen  of  a  cow.  According  to  the  most  trust- 
worthy information,  domestic  and  other  animals  are  not  susceptible 
to  natural  infection  with  typhoid  bacilli,  and  if  the  case  just  mentioned 
was  indeed  one  of  typhoid  it  must  have  been  a  very  exceptional 
occurrence. 

Whether  paratyphoid  bacilli  are  spread  by  milk  is  a  still  unsettled 


486  PATHOGENIC  BACTERIA  IN  MILK 

question,  as  our  knowledge  of  small  epidemics  due  to  this  micro- 
organism is  still  too  fragmentary. 

Cholera  Spirilla. — Three  small  epidemics  of  Asiatic  cholera,  appar- 
ently traceable  to  contamination  of  a  milk  supply,  have  been  reported 
by  Gaffky,  Simpson,  and  Kniippel;  the  spirillum  of  Asiatic  cholera 
itself  has  never,  however,  been  found  in  milk. 

Dysentery  Bacilli. — Dysentery  bacilli  of  the  Shiga,  Flexner,  Kruse,  or 
Strong  type,  which  have  been  isolated  in  cases  of  human  dysentery  in 
adults  and  children,  have  never  been  found  in  milk;  but  since  epidemics 
have  been  traced  to  water,  the  possibility  of  the  occasional  presence 
of  these  bacilli  in  milk  cannot  absolutely  be  denied. 

Bacillus  Diphtherias . — The  Bacillus  diphtherise  has  been  occasionally 
found  in  milk,  and  a  number  of  small  epidemics  of  diphtheritis 
have  been  traced  to  the  milk  supply.  Dean  and  Todd  found  a  case 
of  particular  interest  in  which  4  persons  were  infected  by  the  milk  of 
a  certain  cow  suffering  from  teat  ulcerations  which  contained  diph- 
theria bacilli.  The  ulcerations,  however,  were  not  due  to  the  diphtheria 
bacilli  which  were  present  in  the  open  sores  only  as  an  accidental 
contamination.  Trask,  in  analyzing  the  literature,  found  23  diph- 
theria epidemics  due  to  milk,  15  in  the  United  States  and  8  in  Great 
Britain.  Such  milk  epidemics  usually  show  a  sudden,  almost  explosive 
onset,  because  the  infected  milk  imparts  the  disease  simultaneously 
to  a  number  of  persons.  Milk  is  a  favorable  culture  medium  for  the 
Bacillus  diphtheriae,  which  is  introduced  into  it  through  coughing, 
sneezing,  or  otherwise,  by  persons  engaged  in  collecting  and  handling 
milk,  who  have  more  or  less  recently  passed  through  a  severe,  light, 
or  entirely  masked  and  undiagnosticated  attack  of  the  disease,  and 
who  still  harbor  the  bacilli  in  the  pharynx.  Several  milkborne  diph- 
theria outbreaks  in  California  have  been  reported  by  Ward. 

Scarlet  Fever. — Scarlet  fever  outbreaks,  according  to  Trask,  have 
been  traced  to  milk  51  times.  As  the  cause  of  scarlet  fever  is  still 
unknown  the  statements  as  to  milkborne  epidemics  are  not  very 
trustworthy,  and  it  must  be  remembered  that  the  virus  may  be  spread 
by  those  who  deliver  the  milk  and  in  whom  the  affection  may  be  so 
mild  as  not  to  be  recognized  as  scarlet  fever.  Typical  cases  in  children, 
for  instance,  not  infrequently  lead  to  sore  throats  in  adults,  and  the 
latter,  in  whom  the  infection  is  not  recognized,  may  spread  the 
typical  disease  to  others.  The  author  has  encountered  such  a  case  of 
masked  scarlatina  in  a  nurse  who  spread  the  disease  to  several  persons. 
The  true  state  of  affairs,  so  far  as  the  nurse  herself  was  concerned, 
was  only  accidentally  discovered  by  a  complement  deviation  test.1 
Scarlet  fever  is,  of  course,  not  a  disease  of  cattle,  and  McFadyean's 
experiments  to  produce  the  affection  experimentally  in  calves  were  all 
negative. 

1  For  complement  fixation  test  see  Chapter  VII.     This  is  the  test  used  to  discover  latent 
syphilis  in  persons  (Wassermann  test),  and  it  is  sometimes  also  positive  in  scarlet  fever. 


BACTERIA  OF  MASTITIS  IN  COWS  487 


CATTLE  DISEASES  TRANSMISSIBLE  THROUGH  MILK. 

Foot-and-Mouth  Disease. — In  addition  to  tuberculosis  a  number  of 
other  diseases  of  cattle  may  be  transmitted  through  the  milk  to  man. 
Some  of  these  affections  are  due  to  bacteria,  others  are  due  to  a 
filterable,  invisible  living  virus.  Among  the  latter,  foot-and-mouth 
disease  is  of  importance.  It  is  transmissible  from  afflicted  animals 
to  man  through  raw  milk,  buttermilk,  butter,  cheese,  and  whey.  Man, 
however,  does  not  seem  very  susceptible  to  the  virus  because  in 
European  countries  the  affection  often  occurs  in  widespread  epidemics 
among  cattle,  yet  cases  in  man  are  rare.  The  disease  in  man  may 
take  a  light,  a  serious,  or  even  a  fatal  form.  According  to  Busenius 
and  Siegel,  172  cases  of  hoof-and-mouth  disease  infection  occurred 
in  man  from  1886  to  1896,  of  which  66  could  be  traced  to  milk  and 
1  to  butter.  A  few  cases  transmitted  through  butter  and  1  case 
conveyed  by  soft  cheese  were  reported  after  1896.  It  has  also  been 
proved  by  a  number  of  experiments  that  the  disease,  after  it  has 
been  transmitted  to  man  can  be  retransferred  to  cattle. 

Anthrax. — It  has  been  shown  by  Bollinger,  Feser,  Nocard,  Mc- 
Fadyean  and  others  that  the  milk  of  cows  suffering  from  anthrax 
may  contain  anthrax  bacilli.  Cows  sick  with  anthrax,  however, 
generally  soon  go  dry;  yet  anthrax  bacilli  may  occasionally  be  trans- 
ferred through  milk.  Karlinski  has  reported  the  case  of  a  patient 
convalescent  from  typhoid  fever  who  drank  a  quart  and  a  half  of 
milk  brought  by  a  visitor.  The  patient  shortly  afterward  became 
very  sick  again;  anthrax  bacilli  were  found  in  his  feces,  and  he  died 
after  one  month,  as  the  postmortem  showed,  from  the  intestinal 
form  of  anthrax.  It  was  also  shown  that  the  cow  which  had  furnished 
the  milk  had,  in  the  meantime,  died  from  anthrax.  Bonhoff,  in  the 
examination  of  39  samples  of  butter,  once  accidentally  found  anthrax 
bacilli.  These  reports  show  that  milk  and  milk  products  may 
occasionally,  though  very  rarely,  contain  virulent  anthrax  bacilli. 

Enteritis  in  Cows. — Bacilli  of  the  colon-paratyphoid  group,  causing 
enteritis  in  cows,  may  occasionally  lead  to  intestinal  disturbances  in 
man.  Gaffky  has  reported  a  case  where  the  milk  of  a  cow  suffering 
from  hemorrhagic  enteritis  caused  such  disturbances  in  three  persons. 
Klein  observed  an  epidemic  of  diarrhea  in  London  which  he  thought 
was  due  to  the  Bacillus  enteritidis  sporogenes  infecting  cow's  milk 
from  the  feces  of  these  animals,  but  Hewlett  and  Barton,  who  found 
this  bacillus  in  60  per  cent,  of  the  samples  of  London  market  milk 
examined  for  this  reason  consider  Klein's  claim  as  entirely  unfounded. 

Trembles  and  Malta  Fever. — The  transmission  of  trembles,  or  milk 
sickness,  from  cows  to  man  and  of  Malta  fever  from  goats  to  man 
have  been  discussed  in  Chapters  XXXII  and  XXXIV. 

Bacteria  of  Mastitis  in  Cows. — The  bacteriology  of  inflammation  and 
suppuration  of  the  udder  in  cows  (bovine  mastitis)  is  quite  fully 


488      CATTLE  DISEASES  TRANSMISSIBLE  THROUGH  MILK 

discussed  in  Kitt's  contribution  on  this  subject  to  Kolle  and  Wasser- 
mann's  Manual  on  Pathogenic  Microorganisms.  L.  Frank  was  the 
first  investigator  to  hold  that  mastitis  in  cows  was  generally  due  to  an 
infection,  and,  in  1875,  he  conducted  experiments  showing  that  the 
disease  could  be  produced  by  injecting  fluids  containing  certain 
bacteria  into  the  udder,  and  that  it  could  then  be  again  transferred 
from  diseased  to  healthy  cows.  The  bacteriology  of  bovine  mastitis 
was  subsequently  studied  by  a  large  number  of  authors,  among  whom 
may  be  mentioned  Rivolta,  Dickerhoff,  Mollereau,  Nocard,  Kitt, 
Bang,  Lucet,  Guillebeau,  Jensen,  and  others.  The  disease  may  have 
a  very  rapid  onset  (over  night),  and  it  may  then  become  chronic  or  it 
may  from  the  beginning  have  an  insidious,  slow,  and  chronic  course. 
The  milk,  in  cases  of  very  acute  onset,  shows  marked  changes;  it 
often  contains  coagula,  which  are  sometimes  stained  by  admixture 
with  blood.  In  the  slow,  chronic  cases  not  much  change  is  evident 
in  the  lacteal  fluid.  Sometimes  one-quarter  of  the  udder  alone  is 
affected,  at  other  times  two,  three,  or  all  of  the  four  ventricles.  Some- 
times mastitis  leads  to  grave  general  symptoms,  with  high  fever  and 
prostration.  Recovery  may  occur  in  ten  to  thirty  days,  or  the  disease 
may  extend  over  weeks  and  months;  it  may  make  a  cow  dry  and 
produce  atrophy  or  necrosis  of  the  udder. 

The    bacteria   most    commonly   found    in    suppurative    inflam- 
mations of  the  udder  are:    Bacilli  of  the  colon  group,  first  called 
Bacillus  phlegmasia  uberis  by  Kitt,  now  simply  known  as  colon 
bacillus.     The  organisms   of    this  group   have  already  been  fully 
described.    Streptococci  are  frequently  the  cause  of  mastitis  in  cows, 
but  their  presence  in  milk  is  by  no  means  conclusive  evidence  of  an 
inflammation.    In  fact,  some  of  the  most  common  lactic-acid  bacteria 
in  milk  are  of  the  non-pathogenic  streptococcus  type.    Heinemann, 
however,  has  succeeded,  by  repeated  passages  through  the  bodies  of 
rabbits,  in  changing  non-pathogenic  streptococci  from  milk  into  a 
type  pathogenic  to  rabbits  in  subcutaneous  and  intravenous  injections. 
This,  however,  proves  nothing  as  to  any  original  pathogenicity  when 
the  organisms  are  taken  into  the  stomach  of  man  with  milk.    A  large 
number  of  observers  have  frequently  found  streptococci  in  market 
milk,  as  for  instance  Bergey  (Philadelphia,  50  per  cent,  of  the  samples), 
Eastes  (England,  72.5  per  cent),  Bruening  (Leipzig,  93  per  cent.), 
Conn  and  Esten  (Middle ton,  100  per  cent.)  and  many  others.    Most 
of  the  streptococci  commonly  found  in  milk  belong  to  the  type  of 
Streptococcus  lacticus,  one  of   the  common  lactic-acid  formers  in 
milk,  and  these  cannot  by  any  known  tests,  except  possibly  occasionally 
by  animal  inoculations,  be  distinguished  from  the  Streptococcus  pyo- 
genes.    If  it  is  known  that  a  cow  has  mastitis,  then  the  streptococcus 
present  may  be  of  the  pathogenic,  pyogenes  type;  but  the  disease 
of  the  udder  may  also  be  due  to  bacilli  of  the  colon  group  and  the 
streptococcus  present  be  the  non-pathogenic  lacticus.    The  organism 
known  as  Streptococcus  mastitidis  vaccarum  forms  both  short  and 


NUMBER  AND  SIGNIFICANCE  OF  LEUKOCYTES  IN  MILK     489 

very  long  chains,  and  does  not  liquefy  gelatin.  Some  of  the  stems 
isolated  from  cases  of  bovine  mastitis  form  acid  in  sterile  milk  and 
coagulate  it,  others  do  not  form  acid,  nor  lead  to  coagulation.  In 
considering  the  pathogenicity  to  man  of  streptococci  found  in  cattle, 
it  must  be  remembered  that  the  streptococcus  generally  found  as 
the  cause  of  suppurative  processes  in  cattle  and  known  as  the  Strepto- 
coccus pyogenes  bovis  is  not  fully  identical  with  the  Streptococcus 
pyogenes  found  in  man,  from  which  it  differs  in  certain  cultural  and 
other  characteristics.  (See  chapter  on  Pyogenic  Bacteria  in  Domestic 
Animals.) 

Staphylococci  have  likewise  been  frequently  found  in  inflammation 
of  the  udder  of  the  cow,  and  Guillebeau  has  distinguished  the  follow- 
ing four  types :  Staphylococcus  mastitidis,  Galactococcus  versicolor, 
Galactococcus  flavus,  and  Galactococcus  albus.  Lucet  has  described 
five  different  staphylococci  of  bovine  mastitis,  but  he  has  not  proposed 
special  names  for  them.  Mastitis  in  the  cow  has  been  produced 
experimentally  by  the  injections  of  Bacillus  suipestifer,  Streptococcus 
equi,  Bacillus  avisepticus,  and  Botryococcus  ascoformans. 


NUMBER  AND   SIGNIFICANCE   OF   LEUKOCYTES   IN  MILE. 

Milk  always  contains  certain  cellular  elements.  The  fluid  secreted 
during  the  very  first  stage  of  lactation,  called  colostrum,  shows 
corpuscles  filled  with  fat  granules  known  as  colostrum  corpuscles. 
The  derivation  of  these  bodies  has  been  a  contested  question  for  a 
long  time,  but  the  preponderance  of  evidence  now  appears  to  be  that 
the  majority  of  them  are  mononuclear  leukocytes,  and  only  a  small 
number  are  granular  epithelial  cells,  both  filled  with  fat  granules. 
The  colostrum  corpuscles  disappear  soon  after  the  beginning  of 
lactation,  and  the  most  important  cells  always  found  in  milk  are  the 
leukocytes,  or  white  blood  corpuscles.  Several  authors,  among  those 
who  have  studied  the  significance  of  the  number  of  leukocytes  found 
in  milk  under  various  conditions,  have  very  seriously  discussed  the 
question  how  to  distinguish  in  milk  between  leukocytes  and  pus 
corpuscles.  This  must  appear  somewhat  futile  in  view  of  the  fact 
that  there  is  no  generic  difference  between  a  leukocyte  and  a  pus 
corpuscle.  Histopathologic  investigations  have  shown  beyond  doubt 
that  a  pus  cell  is  nothing  more  or  less  than  a  leukocyte,  which, 
in  consequence  of  inflammatory  stimuli  and  positive  chemotactic 
influences,  has  wandered  out  of  a  bloodvessel  into  the  perivascular 
tissue  or  into  a  preexisting  or  pathologically  formed  cavity.1  In 
inflammatory  conditions  of  the  mammary  glands  an  increase  in  the 
number  of  leukocytes  in  milk  must  be  expected.  It  appears,  on  first, 
thought,  that  the  leukocyte  count  would  aid  in  the  detection  of  milk 
derived  from  a  diseased  udder,  and  indicate  whether  or  not  it  should 

i  This  subject  has  been  fully  discussed  in  the  Chapter  on  Phagocytosis. 


490     NUMBER  AND  SIGNIFICANCE  OF  LEUKOCYTES  IN  MILK 

be  condemned  as  improper  for  use,  repulsive,  and  probably  unwhole- 
some. The  leukocyte  contents  of  wholesome  milk  from  healthy  cows, 
however,  varies  so  much  as  shown  by  more  recent  investigations  based 
upon  accurate  methods,  that  older  standards  resting  upon  inaccurate 
methods  and  upon  insufficient  data  must  be  given  up. 

Methods  of  Estimating  Leukocytes  in  Milk. — STOKES'  METHOD. — 
Place  10  c.c.  of  milk  into  a  centrifuge  tube  and  centrifuge  in  an 
electric  or  other  good  centrifuge  for  ten  minutes.  Draw  off  the  fat 
and  the  clear  fluid  by  the  aid  of  a  pipette  and  leave  only  the  sediment 
in  the  tube.  Spread  a  platinum  loopful  of  the  sediment  over  1  square 
centimeter  on  a  glass  slide  or  cover-glass.  Air  dry,  fix  and  stain  with 
methylene  blue;  wash  in  water,  dry  between  filter  paper  and  mount. 
Count  ten  fields  of  a  one-twelfth  oil-immersion  lens  and  calculate  the 
average  per  field.  It  has  been  customary  on  the  basis  of  this  method 
to  declare  milk  unfit  for  use  when  the  number  of  leukocytes  exceeded 
ten  leukocytes  per  field. 

STEWART'S  METHOD. — Small  glass  tubes  are  used,  closed  at  one 
end  with  a  rubber  stopper.  Place  1  c.c.  of  milk  into  one  of  the  tubes, 
centrifuge  for  ten  minutes.  Then  hold  the  tube  horizontally  and 
draw  out  the  rubber  stopper,  to  which  the  sediment  adheres.  Spread 
the  sediment  over  one  square  centimeter  area  of  a  glass  slide  or 
cover-glass.  Air  dry,  fix  and  stain  with  methylene  blue,  and  count 
the  leukocytes  in  ten  fields  of  a  one- twelfth  oil-immersion  lens.  Milk 
has  been  considered  unfit  for  use  if  the  average  per  microscopic  one- 
twelfth  oil-immersion  field  is  above  twenty- three. 

TROMMSDORF'S  METHOD. — In  this  method  specially  constructed 
centrifuge  tubes  are  used  holding  10  c.c.  and  drawn  out  at  the  bottom 
into  a  fine  caliber.  The  narrow  part  of  the  tube  has  twenty  gradua- 
tions, each  one  representing  0.01  percent,  of  10  c.c.  If  the  sediment, 
therefore,  reaches  to  the  tenth  mark  it  indicates  that  the  10  c.c.  of 
milk  contain  0.1  per  cent,  sediment  or  one  volume  of  the  latter  to  1000 
of  milk.  According  to  Trommsdorf  this  is  the  maximum  amount 
admissible,  and  anything  above  represents  an  excess  in  leukocytes, 
i.  e.,  pus.  While  this  method  simply  indicates  the  percentage  of  sedi- 
ment and  gives  no  information  as  to  its  composition,  it  is  said  to  be  a 
fairly  accurate  and  good  practical  method. 

DOANE-BUCKLEY  METHOD. — This  is  the  most  accurate,  reliable 
procedure,  because  actual  count  of  the  leukocytes  is  made  with  a 
Thoma-Zeiss  counting  chamber  (see  below).  The  method,  as  modified 
by  Campbell  and  described  in  Bulletin  117  of  the  Bureau  of  Animal 
Industry,  includes  the  heating  of  the  milk  to  be  tested,  as  this  separates 
the  leukocytes  from  the  fat  globules,  and  gives  higher  and  more 
accurate  values.  The  effect  of  heating  on  the  leukocyte  count  had 
.been  previously  described  by  Russell  and  Hoffmann.  The  steps  in 
the  Doane-Buckley  method  are  as  follows: 

1.  In  this  as  in  any  other  method  the  sample  examined  should  be 
as  fresh  as  possible,  because  the  formation  of  lactic  acid  favors  the 
precipitation  of  casein,  and  this  interferes  with  obtaining  a  clear 


THOMA-ZEISS  COUNTING  CHAMBER  491 

specimen.  If  milk  must  be  brought  from  a  distance  to  the  laboratory 
it  is  advantageous  to  add  a  few  drops  of  formalin  to  100  c.c.  of  the 
milk  to  prevent  changes  in  reaction.  The  milk  should  be  well  shaken, 
then  10  c.c.  are  filled  into  a  centrifuge  tube.  The  latter  is  now  im- 
mersed in  a  water  bath  at  65°  to  70°  C.  for  ten  minutes,  or  at  80°  to 
85°  C.  for  one  minute.  The  tube,  while  still  warm,  is  at  once  centri- 
fuged  for  ten  minutes. 

2.  Draw  off  5  or  6  c.c.  of  the  fat  and  watery  fluid,  leaving  the 
sediment  undisturbed.  Add  enough  warm  distilled  water  to  make  up 
to  10  c.c.  Shake  well  and  centrifuge  again.  Repeat  this  procedure 
several  times  and  a  clear  sediment  free  from  fat  is  obtained.  Finally, 
draw  off  all  fluid  except  1  c.c.,  which  is  left  in  the  centrifuge  tube. 
Shake  well,  place  a  small  drop  on  a  counting  chamber,  and  count 
several  hundred  squares.  Estimate  the  average  number  of  leukocytes 
per  square  and  from  it  calculate  the  number  of  leukocytes  present  in 
1  cubic  centimeter  of  milk.  Ward  recommends  leaving  only  £  c.c. 
in  the  centrifuge  tube. 

Thoma-Zeiss  Counting  Chamber. — This  instrument,  which  is  used 
in  counting  red  and  white  blood  corpuscles  in  blood,  milk,  pus,  urine, 
and  other  fluid  media,  is  constructed  as  follows :  On  the  centre  of  a 
strong  slide  a  small  round  glass  plate  of  exact  known  thickness  is 
mounted;  a  second  glass  plate,  also  of  known  thickness,  with  a  larger 
circular  opening  in  the  centre  is  so  mounted  on  the  heavy  slide  that 
it  is  outside  of  the  small  round  glass  plate  in  the  centre.  The  outer 
plate  is  exactly  -^  mm.  higher  than  the  small  round  inner  plate, 
which  is  ruled  in  such  a  manner  that  1  square  millimeter  has  been 
divided  into  400  equal  squares.  In  other  words,  each  one  of  the  small 
ruled  squares  is  equal  to  T^7  of  one  square  millimeter.  In  using  the 
counting  chamber  a  small  drop  of  fluid  containing  the  corpuscles  (in 
the  present  case  milk  sediment)  is  placed  on  top  of  the  centre  of 
the  inner  round  (ruled)  glass  plate.  A  clean,  rather  thick  cover- 
glass  is  then  placed  over  the  drop.  This  must  be  done  very  carefully 
in  an  inclined  manner,  to  prevent  air-bubbles  from  entering  between 
the  glass  and  the  drop  of  fluid.  The  cover-glass,  after  being  in  place, 
is  now  (at  the  margin  where  it  rests  on  the  outer  plate)  pressed  down 
with  a  tissue  needle,  glass  rod,  or  lead  pencil.  Some  of  the  fluid 
which  is  between  the  cover-glass  and  the  small,  central,  ruled  glass  plate 
will  run  into  the  moat  formed  between  the  round  inner  and  the  outer 
plate.  The  whole  slide  is  now  lifted  to  the  stage  of  the  microscope  and 
is  focussed  with  a  ^  or  ^  inch  objective.1  A  certain  number  of  leuko- 
cytes, easily  recognizable  by  their  nuclei  and  more  or  less  granular 
protoplasm  are  now  seen.  The  number  of  leukocytes  in  200  squares 
is  counted.  Every  space  seen  through  the  microscope  has  a  base  of 
3-J-Q-  square  millimeter  and  its  height  is  -^  mm.,  which  is  the  distance 
of  the  cover-glass  resting  on  the  outer  plate  from  the  inner  ruled, 

1  As  the  specimen  is  unstained  the  iris  diaphragm  must  be  closed  so  that  the  field  is  only 
dimly  lighted. 


492     NUMBER  AND  SIGNIFICANCE  OF  LEUKOCYTES  IN  MILK 

small,  round,  glass  plate.     Each  of  the  spaces  seen  in  the  field  of 
the  microscope  represents  accordingly  ^nrVir  °^  a  cubic  millimeter. 

If  the  four  hundred  squares  ruled  on  the  inner  plate  were  alike  and 
undivided  into  groups  the  eye  would  easily  lose  track  and  an  accurate 
count  would  be  difficult.  For  this  reason  the  first  one  of  each  group 
of  five  squares  both  from  right  to  left  and  from  above  downward  has 
an  additional  ruling  going  through  the  centre  of  the  square.  These 
lines,  however,  are  ignored  in  counting,  and  their  only  object  is  to 
enable  the  observer  to  see  when  he  has  counted  the  cells  in  five,  ten, 
fifteen,  or  twenty  squares,  or  any  multiple  of  five  squares. 

Calculation  of  the  Result. — Suppose  that  the  sediment  contained 
in  1  c.c.  of  what  was  left  in  the  tube  is  taken  and  that  242  leukocytes 
have  been  counted  in  two  hundred  of  the  squares;  this  would  mean 
an  average  of  1 .21  leukocytes  per  square.  Since  each  square  represents 
3Tnnr  cubic  millimeter,  each  cubic  millimeter  of  the  sediment  would 
contain  1.21  X  4000,  or  4840  leukocytes.  In  blood  work  it  is  customary 
to  indicate  the  number  of  leukocytes  present  in  1  cubic  millimeter  of' 
blood;  but  in  milk,  1  cubic  centimeter  is  used  as  the  standard.  Since 
1  cubic  centimeter  is  equal  to  1000  cubic  millimeters,  it  is  necessary 
to  multiply  4840  by  1000.  This  gives  4,840,000,  which  indicates  the 
number  of  leukocytes  present  in  the  10  c.c.  of  milk,  as  1  c.c.  of 
the  sediment  examined  contained  all  of  the  leukocytes  in  the  10  c.c. 
originally  used.  To  obtain  the  number  of  leukocytes  in  terms  of 
1  c.c.  it  is,  therefore,  necessary  to  divide  4,840,000  by  10,  which  gives 
484,000.  In  other  words,  it  is  found  that  242  leukocytes  present  in 
two  hundred  squares  means  that  the  milk  examined  contained  484,000 
leukocytes  per  1  c.c. 

The  simple  mechanical  rule  for  finding  the  number  of  leukocytes 
is  therefore:  After  repeated  centrifuging,  leave  enough  of  the  clear 
fluid  with  the  sediment  to  make  the  total  of  sediment  and  clear  fluid 
equal  to  1  c.c.  Shake  well;  prepare  counting  chamber;  count  the 
number  of  leukocytes  in  200  squares  and  multiply  the  sum  obtained 

bv  2000  -  (*?°°-XJ°P?) 
oy  zuuu  •  -  V  200  x  10  ' ' 

If  only  0.5  c.c.  was  left  in  the  centrifuge  tube  then  the  sum  of 
leukocytes  found  in  200  squares  is  multiplied  by  1000. 

When  the  leukocytes  are  very  numerous  it  is  sufficient  to  count 
them  in  100  squares  only;  when  they  are  scanty  it  is  better  to  count 
them  in  400  squares.  In  the  former  case  the  sum  must  be  multiplied 
by  4000;  in  the  latter  by  1000,  in  order  to  obtain  the  figures  for  1  c.c. 
of  milk. 

Campbell  has  made  numerous  comparative  tests,  and  he  finds  that 
heating  the  milk  increases  the  leukocytes  in  the  sediment  no  matter 
what  method  of  sedimentation  and  estimation  is  employed.  He  also 
points  out  that  more  recent  knowledge  concerning  the  leukocyte 
contents  of  milk  compels  the  dismissal  of  former  standards  as  unre- 
liable and  inequitable. 

Variability  in  the  Leukocyte  Count  in  Milk  from  Healthy  Cows  — 
Russell  and  Hoffmann  found  that  the  milk  of  some  of  the  cows 


QUESTION^  493 

investigated  was  remarkably  uniform,  while  in  a  considerable  number 
of  cases  the  results  varied  greatly.  The  more  uniform  results  were 
found  in  cows  with  no  previous  history  of  udder  trouble  of  any  kind. 
Some  cows  of  this  kind,  however,  also  showed  great  variations  in 
the  count.  Eighteen  animals  in  a  perfectly  healthy  group  showed 
in  537  tests  a  leukocyte  contents  of  500,000  or  less  in  90  per  cent,  of 
the  cases;  however,  in  16  tests  in  this  group  over  1,000,000  were 
found.  The  authors  state  that  it  is  apparent  from  their  studies  that 
the  leukocyte  content  of  normal  milk  drawn  from  apparently  normal 
animals  is  quite  often  so  high  that  the  milk  would  be  classed  as 
coming  from  diseased  animals  when  judged  by  the  standards  that  have 
heretofore  been  proposed  and  that  therefore  complete  reliance  cannot 
be  placed  upon  quantitative  leukocyte  standards  alone.  Stone  and 
Sprague  made  1167  leukocyte  counts  in  two  perfectly  healthy  cows 
during  a  period  of  about  ten  months,  examining  each  day  both  the 
morning  and  the  evening  milk.  These  counts  varied  from  2,110,000 
to  10,000.  The  highest  counts  of  over  2,000,000  occurred  the  first 
two  days  of  lactation,  but  counts  between  100,000  and  500,000  were 
found  in  29  per  cent,  of  all  counts.  The  authors  state  that  their  con- 
fidence in  an  arbitrary  numerical  leukocyte  standard  as  the  reliable 
criterion  of  the  sanitary  fitness  of  milk  has  been  very  much  shaken; 
but  they  believe  that  the  physiological  average  is  considerably  below 
500,000.  Ward,  likewise,  is  of  the  opinion  that  a  leukocyte  count  alone 
does  not  generally  furnish  data  from  which  the  presence  or  absence 
of  inflammatory  conditions  in  the  udder  can  be  diagnosticated. 


QUESTIONS. 

1.  Discuss  the  presence  of  tubercle  bacilli  in  milk.    In  what  form  of  tuber- 
culosis are  they  most  frequently  found  in  milk? 

2.  In  what  percentage  of  market  milk  have  they  been  found? 

3.  Is  every  milk  containing  tubercle  bacilli  likely  to  produce  tuberculosis 
in  man? 

4.  What  is  the  procedure  for  discovery  whether  samples  of  milk  contain  live 
virulent  tubercle  bacilli  ? 

5.  Give  in  detail  the  method  of  searching  for  tubercle  bacilli  in  butter. 

6.  How  can  typhoid  bacilli  get  into  milk? 

7.  What  is  known  about  milkborne  typhoid  epidemics? 

8.  How  may  the  diphtheria  bacillus  get  into  milk? 

9.  What  is  known  about  milkborne  scarlet  fever  epidemics? 

10.  What  other  cattle  diseases,  aside  from  tuberculosis,  may  be  transmitted 
through  milk? 

11.  What  are  the  most  common  bacteria  in  mastitis  in  cows? 

12.  Discuss  the  number  and  significance  of  leukocytes  in  milk? 

13.  What  is  a  colostrum  corpuscle?    Where  does  it  come  from? 

14.  What  is  Stokes'  method  of  estimating  leukocytes  in  cow's  milk? 

15.  What  is  Stewart's  method? 

16.  What  is  Trommsdorf's  method? 

17.  Which  is  the  most  accurate  method  of  estimating  leukocytes  in  milk? 
Describe  its  details. 

18.  Describe  a  Thoma-Zeiss  blood-corpuscle  counting  chamber. 

19.  Describe  the  method  of  calculating  the  result  of  a  leukocytic  count  in  milk. 

20.  What  effect  has  heating  the  milk  upon  the  leukocyte  count,  and  why? 

21.  Describe  the  variability  of  the  leukocyte  count  in  the  milk  of  normal  cows. 


CHAPTER    XLVIL 

THE  BACTERIOLOGY  AND  THE  BACTERIOLOGIC  EXAMINATION  OF 
MILK  (CONTINUED)— QUANTITATIVE  ESTIMATION  OF  BACTERIA 
IN   MILK— INTERPRETATION   OF  THE   RESULTS   OF   BAC- 
TERIAL  COUNTS   IN   MILK— DETERMINATION  OF  THE 
ACIDITY  OF  MILK— CERTIFIED  MILK— PASTEURIZA- 
TION OF   MILK— ITS    ADVANTAGES    AND 
DISADVANTAGES— STORCH'S  TEST. 

QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK. 

THE  investigations  of  von  Freudenreich,  Henderson,  and  others 
have  shown  that  the  milk  ducts  in  the  udder  of  perfectly  healthy  cows 
contain  numerous  bacteria.  Sedgwick  and  Batchelder,  MacConkey, 
Burr,  von  Freudenreich,  Lux,  and  others  have  found  the  bacteria 
contents  of  milk  freshly  drawn  under  all  possible  aseptic  precautions, 
and  after  the  removal  of  the  foremilk1  and  an  additional  liberal 
amount  of  regular  milk  to  be  from  250  to  1500  per  cubic  centimeter. 
But  these  figures  form  no  basis  for  practical  deductions  when  a 
large  amount  of  milk  is  collected  in  one  pail.  Russell  found  that  the 
mixed  milk  from  a  good  herd,  collected  with  care  and  cleanliness, 
examined  immediately  after  milking,  showed  from  5000  to  20,000 
bacteria  per  cubic  centimeter.  After  milk  has  been  collected  the 
number  of  bacteria  apparently  decreases  for  a  number  of  hours. 
This  is  attributed  to  the  germicidal  property  of  milk.  This  question 
has  recently  been  re-investigated  by  Rosenau  and  McCoy,  who  come 
to  the  following  conclusions: 

"Judged  by  the  number  of  colonies  that  develop  upon  agar  plates, 
the  bacteria  in  milk  first  diminish,  then  increase  in  numbers.  This 
so-called  germicidal  property  of  milk  occurs  only  in  fresh,  raw  fluid. 
For  the  most  part  our  work  plainly  shows  that  no  actual  reduction 
in  the  number  of  bacteria  occurs.  However,  when  compared  with 
the  controls,  a  restraining  action  is  evident.  The  phenomenon, 
therefore,  appears  to  resemble  that  of  a  weak  antiseptic  rather  than 
that  of  a  true  germicide.  When  milk  is  kept  warm  (37°  C.)  the 
decrease  is  pronounced  within  the  first  eight  or  ten  hours.  After  this 
time  the  milk  has  entirely  lost  its  restraining  action.  When  the  milk 
is  kept  cool  (15°  C.)  the  decrease  is  less  marked,  but  more  prolonged. 
The  decrease  in  the  number  of  bacteria  is  largely  apparent,  being  due, 

1  L.  Schulz  found  in  various  specimens  of  foremilk  obtained  from  healthy  udders,  after 
thorough  external  disinfection,  from  50,000  to  79,000  germs  per  cubic  centimeter. 


BEST  METHOD  FOR  ESTIMATING  BACTERIA  IN  MILK     495 

at  least  in  part,  to  agglutination.  The  germicidal  action  of  milk  is 
specific.  This  action  in  milk  and  blood  serum  resemble  each  other 
in  some  particulars,  but  blood  serum  acts  more  quickly  and  much 
more  powerfully  than  milk.  Heating  milk  above  80°  C.  destroys  its 
germicidal  properties.  The  effect  of  lesser  degrees  of  heat  varies 
with  the  microorganism.  Thus  the  restraining  action  for  Bacillus 
lactis  aerogenes  is  weakened  by  first  heating  the  milk  at  55°  C.  and 
almost  destroyed  at  60°  C." 

While  raw  milk,  therefore,  shows  a  diminution  of  bacteria  during 
the  first  hours  after  collection,  a  rapid  multiplication  occurs  later, 
particularly  if  the  milk  has  not  at  once  been  cooled  and  kept  at  a  low 
temperature  permanently  until  used  for  consumption.  If  this  has 
been  done  the  number  of  bacteria  does  not  increase  much  for  thirty- 
six  hours,  but  milk,  according  to  Park,  contains  many  species  of 
bacteria  which  will  multiply  even  at  39°  F.,  i.  e.,  at  a  temperature  not 
much  above  the  freezing  point  of  water.  Park  found  that  a  specimen 
of  milk  containing  originally  3000  bacteria  per  cubic  centimeter,  kept 
at  32°  F.,  showed  a  decrease  of  microorganisms  after  seven  days; 
this  was  also  true  in  a  specimen  containing  originally  30,000  per  cubic 
centimeter.  At  39°  F.  the  counts,  after  seven  days,  showed  4  and  38 
million  respectively;  at  42°  F.,  11  and  120  million;  at  50°  F.,  after  four 
days,  12  and  300  million,  and  at  60°  F.,  after  two  days,  28  and  163 
million.  These  figures  and  those  ascertained  by  various  observers, 
including  Conn,  Esten,  Harrison,  and  others,  demonstrate  the  great 
influence  of  higher  temperatures  upon  bacterial  multiplication  in 
milk.  Since  milk  shipped  to  cities  by  farmers  and  dairies  is  frequently 
insufficiently  cooled,  market  milk  often  shows  a  high  count  even  in 
samples  which  have  been  collected  with  reasonable  care  and  which 
do  not  show  much  dirt  contamination.  Rosenau  found  in  the  milk 
of  Washington,  D.  C.,  an  average  of  22,134,000  bacteria  per  cubic 
centimeter  during  the  summer  of  1906.  Commenting  upon  these 
figures  he  says : 

"So  far  as  numbers  are  concerned  they  need  not  greatly  alarm 
us,  for  we  know  that  disease  is  due  to  agencies  and  conditions  other 
than  merely  the  presence  of  enormous  numbers  of  bacteria.  By 
universal  consent,  however,  milk  containing  excessive  numbers  of 
bacteria  is  unsuitable  for  infant  feeding.  .  .  .  As  we  grow  older 
it  seems  that  the  gastro-intestinal  mucous  membrane  becomes  com- 
paratively immune  or  resistant  to  bacterial  action.  .  .  .  The 
number  of  bacteria  in  milk  is  not  so  important  from  a  public  health 
standpoint  as  the  kind  and  nature  of  the  bacterial  products.  But 
with  cleanliness  and  the  liberal  use  of  ice,  the  total  number  of  bacteria 
can  be  kept  down,  and  this  affords  a  mode  of  protection  against  the 
dangerous  species  and  their  toxic  products.  Milk  containing  few 
bacteria  will  contain  proportionately  few  or  no  harmful  varieties." 

Best  Method  for  Estimating  Bacteria  in  Milk. — The  most  approved 
method  of  estimating  bacteria  in  milk  consists  in  inoculating  a  suitable 


496     QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK 

culture  medium  with  a  definite  amount  of  diluted  milk,  pouring  plates 
and  counting  the  colonies  which  have  developed  after  a  certain  period 
of  time.  While  very  superior  to  making  stains  from  the  sediment  of 
10  c.c.  of  centrifuged  milk,  this  method,  however,  does  not  furnish 
absolutely  true  values.  In  the  first  place  only  live  bacteria  yield 
colonies,  but  as  they  naturally  are  the  chief  concern  in  the  count,  the 
dead  bacteria  are  of  no  great  significance.  Conditions,  however,  in 
which  all  live  bacteria  present  will  develop  on  the  plates  can  never 
be  created,  as  some  microorganisms  are  aerobic,  others  anaerobic,  and 
as  different  species  vary  in  their  optimum  temperatures  of  growth,  in 
their  requirements  as  to  the  exact  reaction  of  the  culture  medium,  etc. 
Even  counts  made  of  various  specimens  of  milk  under  absolutely 
identical  conditions  may  furnish  quite  diverse  results;  for  instance, 
one  sample  may  contain  a  large  number  of  anaerobic  bacteria  and 
another  very  few.  Since  the  plates,  however,  are  prepared  for  aerobic 
growth,  nothing  concerning  the  number  of  anaerobes  is  learned. 
Furthermore,  bacteria  generally  adhere  to  each  other  in  little  groups 
which  cannot  be  entirely  separated  into  individual  microbes  even  by 
energetic  shaking.  Notwithstanding  all  these  defects  the  method  of 
preparing  plates  and  counting  the  colonies  which  have  developed  after 
a  certain  period  gives  a  relatively  accurate  estimate  of  the  bacterial 
content  of  the  milk  at  the  time  when  the  specimen  was  used  in  the 
preparation  of  the  plates. 

Steps  in  the  Quantitative  Bacterial  Analysis  of  Milk. — 1.  Collect  a 
sample  of  milk  under  all  possible  aseptic  precautions  from  the  speci- 
men to  be  examined.  Samples  obtained  from  so-called  loose  milk  in 
a  big  can  must  generally  be  removed  by  the  ordinary  dipper,  but 
should  be  at  once  poured  into  a  sterile  bottle,  the  cork  stopper  or 
cotton  plug  of  which  is  to  be  removed  only  long  enough  to  permit  the 
introduction  of  the  milk  and  then  to  be  at  once  replaced.  Samples 
taken  from  bottles  or  smaller  containers  should  be  procured  with  sterile 
pipettes,  into  which  the  milk  is  drawn  up  by  suction  either  with  a 
rubber  bulb  or  by  the  mouth.  If  the  latter  method  is  used  the  upper 
end  of  the  pipette  must  be  closed  with  a  cotton  plug,  so  that  no  trace 
of  saliva  can  run  into  the  pipette.  In  general,  however,  this  method, 
even  when  the  pipette  is  protected,  is  not  to  be  recommended.  In 
the  examination  of  certified  milk  it  is  best  to  take  one  of  the  original 
small  bottles  without  opening  it.  In  whatever  manner  the  sample  is 
procured  it  must  immediately  be  packed  in  ice  to  avoid  the  danger 
of  a  great  multiplication  of  bacteria  during  the  period  of  time  elapsing 
between  the  collection  of  the  specimen  and  its  inoculation  into  culture 
media  in  the  laboratory.  The  can  or  bottle  from  which  the  specimen 
is  taken  should  first  be  well  shaken  and  agitated.  It  is  also  well  to 
take  the  temperature  of  the  milk  finally,  because  a  large  number  of 
bacteria  in  cold  milk  would  generally  indicate  improper  collection, 
while  a  large  number  in  warmer  milk  may  simply  point  to  a  multi- 
plication of  lactic-acid  bacteria  in  milk  collected  cleanly.  After 


STEPS  IN  QUANTITATIVE  BACTERIAL  ANALYSIS  OF  MILK   497 

thoroughly  shaking  the  original  bottle  the  sample  from  certified 
milk  can  best  be  procured  at  the  laboratory  by  perforating  the  card- 
board cover  with  a  knife  sterilized  over  a  flame  of  a  Bunsen  burner 
and  inserting  the  sterile  pipette  through  the  hole.  In  this  way  all 
danger  of  increasing  the  bacterial  contents  of  the  milk  during  manipu- 
lation is  entirely  avoided.  Samples  should  be  taken  in  quantities  of 
not  less  than  10  c.c.  because  of  the  need  for  duplicates,  controls,  etc. 
The  sterile  pipettes  used  in  the  collection  of  samples  outside  of  the 
laboratory  should  be  carried  in  metal  boxes  or  in  sterilized  glass  tubes. 
To  use  a  single  pipette  for  collecting  a  number  of  samples  and  steril- 
izing it  between  samples  by  dipping  into  sulphuric  acid  and  then 
into  sterile  water  is  not  a  good  practice. 

2.  Milk  contains  too  many  bacteria  to  allow  it  to  be  mixed  in 
quantities  of  1  c.c.  with  the  culture  media  employed,  hence  it  must  be 
diluted  with  sterile  water.    The  water  can  be  kept  in  ordinary  glass 
bottles.    If  volumetric  flasks  are  used  considerable  space  should  be 
left  over  the  10  c.c.  or  100  c.c.  mark,  so  that  the  dilute  fluid  can  be 
well  shaken.    The  dilutions  used  are  1  in  10,1  1  in  100,  1  in  1000, 
1  in  10,000,  1  in  100,000,  and  1  in  1,000,000.    For  the  examination 
of  market  milk  the  dilutions  are  generally  1  in  1000,  1  in  10,000,  and 
1  in  100,000.    Sterilize  9  c.c.  and  99  c.c.  of  ordinary  clean  tap  water 
in  bottles  closed  with  cotton  plugs.    As  a  rule,  if  100  c.c.  are  placed  in 
bottles  and  sterilized  in  the  autoclave  for  a  sufficient  time  the  water 
will  be  reduced  by  evaporation  to  about  99  c.c.    Preliminary  tests, 
however,  must  determine  how  much  water  should  be  taken  to  be 
exactly  reduced  to  9  c.c.  after  the  sterilization.     The  dilutions  are 
made  in  the  following  manner:    Add  1  c.c.  of  the  milk  which  has 
been  well  shaken,  with  a  sterile  graduated  pipette  to  the  bottle  con- 
taining 9  c.c.  and  also  1  c.c.  to  the  bottle  containing  99  c.c.  of  sterile 
water;  shake  well  for  several  minutes,  taking  care  that  the  fluid  does 
not  come  in  contact  with  the  cotton  plug.    This  gives  the  dilutions 
of  1  in  10  and  1  in  100.    From  these  the  dilutions  of  1  in  1000  and  1  in 
10,000  can  be  prepared  and  from  these  again  the  dilutions  of  1  in 
100,000  and  1  in  1,000,000. 

3.  Before  the  dilutions  are  made  the  culture  media  must  be  pre- 
pared.    The  medium  generally  used  is  10  c.c.  of  an  agar  medium 
containing  1  per  cent,  of  agar,  with  a  reaction  of  1.5  per  cent,  acid  to 
phenolphthalein.     The  medium  should  be  melted  in  a  water  bath, 
then  cooled  down  to  45°  C.    To  control  the  temperature  exactly  a 
thermometer  should  be  placed  in  one  of   the   agar  tubes   in   the 
water  bath.     When  it  registers  45°  it  indicates  that  this  is  also  the 
temperature  of  the  agar  in  the  other  tubes.    The  tube  containing  the 
thermometer  is,  of  course,  not  used  for  pouring  plates. 

4.  When  the  media  are  melted  and  of  the  proper  temperature, 
place  with  sterile  pipettes  1  c.c.  of  the  dilutions  (for  example,  those  of 

1  Only  applicable  in  the  very  best  forms  of  certified  milk. 
32 


498     QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK 

1  in  10(30,  1  in  10,000,  and  1  in  100,000)  into  the  lower  portions  of 
Petri  dishes.1  Then  add  the  melted  agar;  mix  well  with  the  1  c.c. 
of  diluted  milk  in  the  Petri  dish  by  properly  moving  and  shaking. 
This  should  be  done  carefully  so  that  the  medium  does  not  run  over. 

5.  As  soon  as  the  agar  has  again  become  solid  the  Petri  dishes  are 
inverted,  the  now  upper  portion  with  the  solid  medium  in  it  is  lifted 
away  from  the  lower  portion.    Into  the  latter  a  piece  of  filter  paper 
with  a  drop  of  glycerin  on  it  is  placed.    This  arrangement  insures 
against  moisture  collecting  on  the  agar  and  spoiling  the  count  by 
spreading  the  colonies  in  a  diffuse  manner.    The  Petri  dish  is  now 
placed  in  an  inverted  position  (culture  medium  above,  plate  with 
filter  paper  below)  into  the  incubator,  and  is  kept  there  at  37°  C.  for 
forty-eight  hours.     Another  method  recommended  to  prevent   the 
collection  of  moisture  on  the  agar  is  to  use  a  porous  earthenware 
cover  for  the  Petri  dish.    The  former  method,  however,  is  preferable 
to  the  use  of  a  non-transparent  cover. 

6.  After  forty-eight  hours  the  colonies  on  and  in  the  depth  of  the 
agar  are  counted  in  the  manner  described  in  Chapter  XIV,  p.  174.    It 
is  well,  however,  to  count  all  the  colonies  which  have  developed  and  not 
merely  a  number  in  a  portion  of  the  agar.    A  so-called  blank  control 
should  be  made  for  each  set  of  specimens.     This  is  done  in  the  follow- 
ing manner:    One  c.c.  of  the  sterile  water  to  which  no  milk  whatever 
has  been  added  is  poured  into  a  Petri  dish  and  then  the  melted  agar 
is  added.    The  plate  is  incubated  with  the  others  and  examined  after 
forty-eight  hours.     It  should  be  entirely  sterile,  or  it  may  perhaps 
have  developed  one  or  two  colonies  on  the  surface,  which  might 
possibly  be  due  to  air  contamination  during  manipulation.     The 
count  is  made  on  those  plates  which  have  developed  between  200 
and  400  colonies.    This  is  considered  to  be  the  dilution  which  gives 
the  most  trustworthy  count  from  which   to  calculate  the  bacterial 
contents  of  the  milk. 

Heinemann  and  Glenn  have  made  some  experiments  to  determine 
whether  it  is  preferable,  in  order  to  get  an  exact  count,  to  incubate 
at  20°  C.  or  at  37°  C.  They  found  that  in  dextrose-litmus-agar2 
the  number  of  colonies  after  one  day  is  larger  at  37°  C.;  after  two 
days  the  number  is  higher  at  20°  C.,  and  after  three  days  the  number 
of  colonies  at  20°  C.  is  about  double  that  at  37°  C.  In  lactose  agar 
the  conditions  are  practically  the  same.  There  are  no  acid  colonies 
in  either  dextrose  or  lactose  agar  after  twenty-four  hours  at  20°  C. 
After  two  days  the  number  of  acid  colonies  in  both  dextrose  and  lactose 
agar  is  considerably  larger  at  37°  C.  than  at  20°  C.,  but  after  three 
days  the  proportion  is  reversed,  as  the  acid  colonies  develop  more 
rapidly  after  two  days  at  20°  than  at  37°  C.  The  proportionate  rate  of 

1  Two  Petri  dishes  should  be  prepared  from  each  dilution. 

1  The  culture  media  used  in  these  tests  were  prepared  as  follows :  A  sterile  litmus  solution 
of  Merck's  pure  extract  of  litmus  was  poured  into  a  Petri  dish,  then  the  dilute  milk  was  added, 
and  finally  the  melted  dextrose  or  lactose  agar  was  poured  into  the  dish. 


THE  RESULTS  OF  BACTERIAL  COUNTS  IN  MILK         499 

acid  colonies,  however,  compared  with  the  total  colonies  developed  is 
smaller  at  20°  C.  than  at  37°  C.  Heinemann  and  Glenn,  therefore, 
favor  an  incubation  at  20°  C.  for  three  days  over  one  at  37°  C.  for  two 
days,  and  they  think  that  dextrose-litmus-agar  is  better  than  lactose- 
litmus-agar,  because  more  acid  producers  develop  on  the  former  than 
on  the  latter.  For  incubation  at  20°  C.  they  used  an  ice-chest  kept 
cool  by  circulating  tap  water  and  heated  by  an  incandescent  electric 
light  connected  with  a  thermoregulator,  which  disconnected  the  current 
whenever  the  temperature  rose  above  20°  C. 

Interpretation  of  the  Results  of  Bacterial  Counts  in  Milk. — The 
question  of  the  number  of  bacteria  found  in  milk  is,  of  course,  not 
alone  of  theoretical  but  also  of  great  practical  interest.  Milk  con- 
taining a  great  number  of  bacteria  may  be  unwholesome,  and  in  that 
case  should  be  condemned  and  exluded  from  use  as  a  food.  Several 
cities  in  the  United  States  have  fixed  an  arbitrary  standard  of  maxi- 
mum count  beyond  which  milk  shall  be  condemned.  It  appears, 
however,  that  the  consensus  of  opinion  among  authorities  on  the 
question  of  milk  hygiene  is  that  milk  cannot  be  judged  from  a  mere 
bacterial  count  as  to  its  fitness  or  non-fitness  for  human  consumption. 
Rosenau's  opinion  as  to  the  significance  of  large  numbers  of  bacteria 
in  milk  has  already  been  given;  others  have  expressed  similar  or 
even  more  pronounced  views. 

Rodgers  and  Ayers,  for  example,  state:  "Numerical  bacteriological 
standards  which  are  unquestionably  of  value  are  necessarily  arbitrary, 
and  are  based  on  the  count  of  total  bacteria  only.  Special  methods 
are  necessary  to  obtain  any  insight  into  the  relative  numbers  of 
bacteria  of  the  different  groups  occurring  in  milk,  and  by  the  infor- 
mation thus  obtained  to  form  an  opinion  regarding  the  cleanliness 
and  care  observed  in  producing  and  marketing  the  milk.  ...  A 
count  of  the  total  bacteria  does  not  always  give  a  true  indication  of  the 
conditions  under  which  milk  is  produced.  In  order  to  interpret  results 
intelligently  it  is  necessary  to  know,  if  possible,  the  age  of  the  milk 
and  the  temperature  at  which  it  has  been  held.  Clean  milk  which 
has  been  held  several  hours  in  a  warm  place  may  contain  more 
bacteria  than  dirty  milk  when  fresh  or  even  after  two  or  three  days 
if  it  has  been  held  at  a  low  temperature." 

Swithinbank  and  Newman,  after  giving  figures  of  bacteria  found 
in  milk  in  various  cities,  say:  "Many  similar  investigations  with 
very  similar  results  might  be  quoted,  but  the  above  will  suffice  to 
convey  an  impression  of  the  bacterial  contents  of  many  milks.  It  is, 
of  course,  needless  to  add  that  quantitative  records,  whether  repre- 
sented by  high  or  low  figures,  are  in  no  sense  an  exact  index  as  to  the 
injurious  nature  or  otherwise  of  the  milk  in  question,  or  as  to  its 
value  for  human  consumption.  A  knowledge  of  the  exact  quality  of 
the  milk,  of  the  kind  of  organisms  and  their  role,  is  necessary  before 
any  valid  conclusions  can  be  drawn.  .  .  .  The  fact  is  that  numeri- 
cal estimation  of  organisms  is  not,  by  itself,  a  sufficient  criterion. 


500     QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK 

All  the  circumstances  must  be  taken  into  consideration,  including  the 
condition  of  the  farms,  the  presence  of  preservatives,  and  the  species 
of  the  bacteria." 

Weigmann,  in  Sommerf eld's  Manual  on  Milk,  says :  "Reports  as 
to  the  number  of  germs  in  milk  naturally  are  very  variable;  they 
simply  indicate  up  to  the  present  time  that  the  bacterial  contents  are 
occasionally  very  high.  This  in  most  cases  is  not  of  as  bad  a  signifi- 
cance as  the  figures  make  it  appear.  However,  such  figures  gener- 
ally point  to  an  unclean  method  of  obtaining  the  milk,  and  this  will 
betray  itself  generally  by  the  presence  of  a  larger  amount  of  dirt  or 
they  point  to  a  not  very  careful  or  not  rational  method  of  treating 
the  milk." 

Conn's  opinion  is  expressed  as  follows:  "It  is  probably  impossible 
to  fix  upon  any  standard  as  to  the  number  of  bacteria  which  whole- 
some milk  may  contain.  Should  we  condemn  milk  when  it  has  10,000 
per  c.c.  or  30,000  or  1,000,000  bacteria?  To  fix  a  standard  is  difficult, 
because  the  number  is  so  dependent  upon  the  temperature  and  the 
season  of  the  year.  .  .  .  Sometimes  this  number  (of  bacteria  in 
special  milk)  has  been  fixed  at  10,000,  in  other  cases  at  30,000.  This 
is  practicable  for  small  dairies  where  the  dealer  wishes  to  furnish 
a  special  product  at  a  special  price,  and  where  the  dairy  is  within 
a  short  distance  of  the  consumer.  .  .  .  But  for  the  general 
milk  supply  of  a  large  city  it  has,  up  to  the  present  time,  been  found 
quite  impracticable  to  suggest  any  bacteriological  standard  without 
excluding  too  large  a  portion  of  the  milk  which  will  be  brought  into 
the  city.  Moreover,  it  seems  by  no  means  sure  that  such  a  standard 
would  be  of  much  practical  value,  because  even  though  the  number 
be  large  the  milk  may  be  perfectly  wholesome  if  they  are  of  the 
normal  lactic  type;  whereas  a  much  smaller  number  of  bacteria  in 
another  sample  of  milk  might  make  it  decidedly  injurious  if  the 
bacteria  should  be  of  a  different  character. 

"These  various  facts  raise  the  question  whether  a  bacteriological 
analysis  which  shall  differentiate  the  different  kinds  of  bacteria  from 
each  other  is  possible  and  practical.  Is  it  possible  to  devise  some 
means  of  analysis  of  the  bacteria  in  milk  which  shall  give  the  numbers 
of  the  different  kinds  of  bacteria,  separating  the  normal  forms  from 
those  that  render  the  milk  suspicious?  If  we  could  do  this,  the 
practical  analysis  of  city  milk  might  be  more  useful  and  might  become 
an  efficient  means  in  the  hands  of  boards  of  health  in  protecting  the 
public  from  the  dangers  in  its  milk  supply.  There  has  hitherto  been 
no  attempt  made  to  develop  such  a  method  of  differential  analysis  of 
milk,  and,  indeed,  at  the  present  time  we  know  too  little  in  regard  to 
the  relations  of  the  different  species  of  bacteria  to  the  wholesomeness 
of  milk  to  make  an  analysis  absolutely  reliable." 

Jensen  expresses  himself  as  follows  concerning  this  question: 
"Of  course,  the  number  of  bacteria  in  market  milk  varies  greatly 
according  to  its  care  and  to  the  temperature  of  the  air.  Experience 


THE  RESULTS  OF  BACTERIAL  COUNTS  IN  MILK         501 

gained  in  most  of  the  larger  cities  shows  the  bacterial  content  of 
market  milk  to  be  seldom  below  50,000  to  100,000  per  c.c.,  but  it 
is  often  greater,  varying  between  1,000,000  to  30,000,000;  indeed, 
not  infrequently,  even  from  100,000,000  to  150,000,000  have  been 
found,  and  such  milk  may  not  be  noticeably  tainted.  .  .  .  It  is 
known  that  sour  milk  has  no  harmful  effect  on  healthy  people.  But 
it  is  -different  with  those  suffering  with  catarrh  of  the  stomach,  and 
even  with  small  children.  .  .  .  The  number  of  bacteria  in  milk 
does  not  give  us  a  safe  criterion  in  this  connection,  but  the  degree  of 
acidity  furnishes  a  reliable  guide." 

Sommerfeld's  Manual  on  Milk  contains  the  laws  governing  the 
milk  supply  of  the  German  Empire  and  of  a  number  of  the  states 
forming  it,  as  well  as  the  ordinances,  rules,  and  regulations  of  two 
hundred  cities.  Yet  not  a  word  is  found  anywhere  in  them  about  a 
limit  of  a  permissible  bacterial  count,  while  inspections  of  the  cows  and 
dairies  by  competent  veterinarians  and  detailed  rules  as  to  cleanli- 
ness, handling,  cooling,  bottling,  labelling,  and  selling  of  the  milk  are 
amply  provided. 

It  is  quite  evident  that  there  should  be  no  ironclad  rule  as  to  the 
number  of  bacteria  permissible  in  ordinary  market  milk.  No  com- 
petent investigator  has  ever  claimed  that  a  milk  containing  1,000,000 
bacteria,  provided  that  they  are  of  the  ordinary  saprophytic  kind, 
normally  found  in  milk,  is  unwholesome  to  older  children  or  adults, 
and  it  has  not  even  been  shown  that  such  a  milk  is  unwholesome  to 
infants.  It  is,  of  course,  different  with  the  dirty  milk  containing  many 
millions  of  bacteria  as  sold  in  summer  in  large  cities  throughout  the 
world.  If  it  cannot  be  shown  that  every  milk  containing  1,000,000 
bacteria  is  unwholesome  to  man,  why  should  there  be  an  ordinance 
condemning  such  milk?  The  health  authorities  of  communities 
should  make  bacterial  counts  of  milk  regularly,  because  high  counts 
often  indicate  improper  handling,  unclean  dairies,  or  cows  sick  from 
udder  affections.  The  knowledge  gained  from  bacterial  counts  should 
be  used  to  discover  such  unhygienic  conditions,  and  they  should  be 
corrected.  But  to  condemn  a  milk  solely  upon  the  ground  that  it 
contains  a  certain  number  of  bacteria  without  any  further  informa- 
tion about  it  is  an  inequitable,  unjust  measure  which  may  lead  to 
the  unnecessary  destruction  of  a  wholesome  food.  Metchnikoff  and 
other  investigators  have  for  several  years  advocated  the  use  of  milk 
soured  by  lactic-acid  bacteria  as  one  of  the  best  means  to  prevent 
unwholesome  fermentations  in  the  human  intestines  and  as  one  of  the 
most  important  means  to  prolong  human  life.  Cultures  of  lactic-acid 
bacteria  have  also  been  used  as  sprays  in  certain  pathologic  condi- 
tions of  the  nasopharyngeal  mucous  membranes.  So  milk  containing 
millions  of  lactic-acid  bacteria  and  a  corresponding  amount  of  lactic 
acid  is  not  only  not  unwholesome,  but  probably  (at  least,  according 
to  Metchnikoff  and  his  followers)  a  food  particularly  well  adapted  to 
promote  longevity.  Milk,  however,  which  has  already  attained  a 


502     QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN  MILK 

higher  degree  of  acidity  should  not  be  permitted  to  be  sold  as  fresh, 
sweet  milk,  and  health  authorities  should  include  the  determination 
of  acidity  among  the  measures  governing  the  sale  of  milk. 

Determination  of  the  Acidity  of  Milk. — The  acidity  of  milk  can  be 
determined  as  follows:  Place  50  c.c.  of  milk  into  a  beaker  and  add 
2  c.c.  of  a  2  per  cent,  alcoholic  solution  of  phenolphthalein  as  an 
indicator.  Then  add  from  a  burette  (gradually  under  constant  shaking 
or  stirring  with  a  glass  rod)  decinormal  solution  of  sodium  hydroxide 
(^  sol.  NaHO)  until  all  acid  has  been  neutralized  and  the  fluid 
retains  a  very  faint  pink  color.  This,  of  course,  means  that  the 
phenolphthalein  in  solution  now  indicates  that  all  acid  has  been 
neutralized  and  that  a  trace  of  alkaline  decinormal  solution  of  sodium 
hydroxide  has  been  added  in  excess.  Each  cubic  centimeter  of  -^  sol. 
NaHO  is  equivalent  to  0.009  gram  of  lactic  acid. 

In  order  to  obtain  the  amount  of  acidity  in  50  c.c.  of  milk  each 
cubic  centimeter  of  decinormal  solution  used  out  of  the  burette  to 
effect  complete  neutralization  must  be  multiplied  by  0.009.  The 
product  should  again  be  multiplied  by  2  in  order  to  obtain  the  amount 
of  acidity  per  100  c.c.  of  milk.1  The  rule,  therefore,  when  50  c.c. 
of  milk  have  been  used  is  simply:  "Multiply  the  number  of  cubic 
centimeters  of  -^  sol.  NaHO  used  out  of  the  burette  by  0.018;  the 
result  will  be  the  percentage  of  acidity  in  milk."  In  this  calculation 
the  total  acidity  is  expressed  as  lactic  acid;  part  of  the  acidity  may  be 
due  to  other  fatty  acids,  such  as  butyric,  succinic,  formic,  acetic,  but 
this  is  immaterial,  because  for  practical  purposes  it  is  quite  sufficient 
to  know  the  total  acidity. 

For  example,  in  the  test  of  50  c.c.  of  milk,  9.3  c.c.  of  -^  sol.  NaHO 
out  of  the  burette  have  been  used  in  order  to  accomplish  complete 
neutralization;  the  result  then  is  9.3  X  0.018  =  0.167  per  cent,  of 
acid  in  the  milk  tested.  It  is  generally  held  that  market  milk  should 
not  show  more  than  0.2  per  cent,  of  total  acidity,  because  any  excess, 
as  a  rule,  indicates  that  lactic-acid  fermentation  has  pretty  well  begun. 

Recording  of  Results  of  Bacterial  Counts  in  Round  Numbers. — As  a 
rule,  bacterial  counts  of  milk  are  expressed  from  the  number  of 
colonies  counted  on  the  plates,  in  round  numbers,  so  that: 

Counts  below  100,000  are  rounded  off  in  terms  of  10,000;  for 
instance,  20,000. 

Counts  between  100,000  and  500,000  are  rounded  off  in  terms  of 
50,000;  for  instance,  350,000. 

Counts  between  500,000  and  1,000,000  in  terms  of  100,000;  for 
instance,  700,000. 

Counts  between  1,000,000  and  2,000,000  in  terms  of  200,000;  for 
instance,  1,400,000. 

Counts  between  2,000,000  and  5,000,000  in  terms  of  500,000;  for 
instance,  2,500,000. 

1  It  is,  of  course,  customary  to  express  the  acidity  in  per  cent.,  i.  e.,  for  100  c.c.  of  milk. 


CERTIFIED  MILK 


503 


Counts  above  5,000,000  are  expressed  in  round  millions.    The 
following  figures,  therefore,  are  used. 


Below 
Above 


10,000 

10,000 

20,000 

30,000 

40,000 

50,000 

60,000 

70,000 

80,000 

90,000 

100,000 

150,000 

200,000 

250,000 

300,000 

350,000 

400,000 

450,000 

500,000 

600,000 


Above 


700,000 
800,000 
900,000 
1,000,000 
1,200,000 
1,400,000 
1,600,000 
1,800,000 
2,000,000 
2,500,000 
3,000,000 
3,500,000 
4,000,000 
4,500,000 
5,000,000 
6,000,000 
7,000,000 
8,000,000 
9,000,000 
10,000,000 


Counts  on  certified  milk  should  be  made  and  expressed  as  exactly 
as  possible;  so  that  if  a  count  on  a  plate  from  a  milk  diluted  ten 
times  has  been  made  and  shows  321  colonies  the  figure  given  is 
3210  per  1  c.c.  of  milk. 

CERTIFIED  MILK. 

By  certified  milk  is  understood  a  milk  produced  under  the  super- 
vision of  a  Medical  Milk  Commission  which  has  established  a  certain 
standard  to  which  the  milk  must  conform,  and  which  has  issued  a 
set  of  rules  according  to  which  the  dairy  furnishing  the  milk  must 
be  conducted.  These  rules  generally  include  the  following  points: 
The  stables  of  the  dairy  must  be  thoroughly  hygienic,  the  drainage 
perfect,  and  the  water  supply  first  class.  The  stock  should  be  subjected 
at  regular  intervals  to  the  tuberculin  test  and  be  under  the  almost 
constant  supervision  of  a  competent  veterinarian.  All  sick  or  sus- 
picious cows  should  immediately  be  removed  from  the  herd  and  new 
stock,  before  being  allowed  to  enter,  thoroughly  examined.  The 
milk  should  be  drawn  by  perfectly  clean  milkers  who  are  free  from 
disease  themselves.  If  smallpox,  typhoid  fever,  diphtheria,  scarlet 
fever,  measles,  and  other  contagious  diseases  occur  in  the  vicinity  of 
the  dairy  a  strict  supervision  should  be  established  and  the  milkers  not 
allowed  to  come  into  contact  with  persons  sick  with  these  diseases,  nor 
may  they  enter  places  in  which  such  diseases  exist.  The  hands  of  the 
milker  and  the  udder  of  the  cow  should  be  cleansed  before  milking 
and  the  milk  received  into  sterile  receptacles,  strained  through  a  fine 
wire  gauze,  and  a  layer  of  absorbent  cotton,  distributed  to  sterile 
bottles  and  cooled  down  at  once  to  50°  F.  or  lower.  The  bottles  must 
be  sealed  in  a  manner  preventing  subsequent  contamination,  kept 


504  THE  PASTEURIZATION  OF  MILK 

cool,  packed  in  ice  during  transportation  except  in  winter,  and  reach 
the  consumer  within  thirty  hours  after  being  drawn. 

Milk  commissions  generally  employ  a  veterinarian,  a  bacteriologist, 
and  a  chemist  as  experts  for  the  control  of  the  animals  kept  in  the 
dairy  and  of  the  milk  furnished  by  them.  "The  duties  of  the  veter- 
inarian," as  defined  by  Ward,  one  of  the  foremost  American  experts 
on  milk,  "are  to  determine  the  general  health  of  the  animals,  to 
observe  the  sanitary  conditions,  and  to  scrutinize  the  technique  of 
milk  handling.  In  general,  his  duty  is  to  determine  if  the  conditions 
of  the  agreement  of  the  dairyman  with  the  commission  are  being 
observed.  His  criticism  and  suggestions  must  maintain  that  degree 
of  alertness  on  the  part  of  the  foreman  of  milkers  and  other  employees 
that  shall  minimize  the  possibility  of  contamination  of  the  milk.  The 
control  of  bovine  tuberculosis  is  a  task  that  demands  the  utmost 
vigilance.  Without  care  in  regard  to  this  disease  the  pretentious 
of  a  certified  dairy  are  fraudulent.  When  not  vigorously  dealt  with 
it  constitutes  the  greatest  menace  to  the  financial  success  of  a  certified 
dairy.  Tuberculin  tests  a  year  apart,  with  careless  supervision  of 
additions  to  the  herd,  are  useless  in  a  herd  that  was  badly  infected 
at  the  beginning,  for  tuberculosis  will  keep  pace  with  lax  efforts 
directed  against  it.  It  is  not  sufficient  to  test  merely  the  cows  that 
happen  to  be  in  milk  at  the  time  of  the  test.  Every  dry  cow  should 
be  included.  In  an  infected  herd  a  test  once  in  six  months  is  regarded 
as  necessary,  followed  each  time  by  thorough  disinfection  of  the  stable. 
The  control  of  tuberculosis  cannot  be  accomplished  by  one  test, 
carried  out  in  a  perfunctory  manner,  but  the  struggle  must  extend 
over  years.  Additions  to  the  herd  must  be  tested  with  tuberculin, 
but  there  is  always  danger  that  an  animal  though  not  reacting  may 
introduce  the  disease.  On  this  account  it  is  far  better  to  subject 
each  animal  added  to  the  herd  to  a  three  months'  quarantine  with  a 
tuberculin  test  at  the  beginning  and  end  of  this  period.  During  the 
period  the  milk  may  be  used." 

Milk  sold  as  certified  under  the  supervision  of  a  medical  milk 
commission  should  be  examined  about  once  a  week  by  a  competent 
bacteriologist  and  its  bacterial  content  should  not  be  above  10,000. 
This  expensive  milk  is  almost  exclusively  used  for  the  feeding  of 
infants,  and  those  paying  a  high  price  should  have  full  assurance  that 
they  get  as  excellent  an  article  of  food  as  they  have  a  right  to  expect. 
In  the  State  of  New  Jersey,  where  the  movement  creating  medical  milk 
commissions  originated  under  the  leadership  of  Henry  L.  Coit,  of 
Newark,  special  laws  have  been  enacted  to  protect  the  sale  of  certified 
milk  against  any  product  not  coming  up  to  the  proper  standard. 

THE  PASTEURIZATION  OF  MILK. 

As  previously  explained,  sterilization  consists  in  exposing  an  object 
to  such  (generally  thermal)  influences  that  all  life  in  it  is  destroyed 


THE  PASTEURIZATION  OF  MILK  505 

and  that  no  fermentative,  putrefactive,  or  similar  processes  can 
occur  in  it.  It  has  been  shown  how  bacterial  culture  media  are 
sterilized  so  that  they  may  be  used  for  the  development  of  pure 
cultures.  Organic  material  in  general  and  certain  foodstuffs,  par- 
ticularly meat  and  milk,  are  excellent  soils  for  the  development  of  a 
host  of  microorganisms.  Their  growth  may  so  change  foodstuffs  that 
they  become  unfit  for  food  both  on  account  of  features  repulsive  to 
our  senses  and  because  they  may  actually  contain  dangerous  poisons. 
It  has  been  long  known  that  low  temperatures  largely  prevent  putre- 
factive processes,  and  it  had  also  been  observed  that  high  temperatures 
may  be  used  for  the  same  purpose.  The  Japanese  have,  for  a  long 
time  been  in  the  habit  of  heating  their  rice  wine  or  sake  in  spring  to 
preserve  it  during  the  summer.  When  Pasteur  studied  the  changes  in 
wine  and  beer,  known  as  the  diseases  of  wine  and  beer,  due  to  certain 
microorganisms  which  develop  subsequent  to  the  alcoholic  fermenta- 
tion of  the  yeast  cells,  or  saccharomyces,  he  tried  to  devise  a  means 
of  checking  such  undesirable  growth.  As  he  had  finally  successfully 
shattered  the  old  ideas  of  spontaneous  generation,  and  demonstrated 
the  requirements  of  reliable,  absolute  sterilization,  the  latter  pro- 
cedure at  once  suggested  itself.  It  was,  however,  soon  found  that 
sterilization  could  not  be  employed  to  protect  wine  or  beer  against 
undesirable  microbic  multiplication  and  changes,  because  it  destroyed 
certain  valuable  properties  in  these  beverages  and  was  too  expensive 
on  account  of  the  excessive  breakage  of  closed  filled  bottles  exposed 
for  a  considerable  time  to  the  action  of  the  temperature  of  boiling 
water  or  steam.  Pasteur  then  devised  methods  of  using  temperatures 
considerably  below  the  boiling  point  for  certain  periods  of  time, 
which  while  not  producing  absolute  sterilization,  killed  most  micro- 
organisms and  produced  conditions  under  which  articles  of  food 
acquired  more  stable  keeping  qualities.  This  process  is  now  generally 
known  as  pasteurization.  After  medical  bacteriology  had  become 
firmly  established  by  the  work  of  Robert  Koch  the  dangers  which 
might  lurk  in  infected  milk  were  not  only  clearly  recognized,  but 
were  for  a  time  much  overestimated,  and  an  agitation  for  the  general 
sterilization  of  milk  resulted.  At  one  period  a  great  quantity  of  the 
cow's  milk  fed  particularly  to  infants  and  children,  but  also  to  adults, 
was  sterilized  by  being  boiled,  often  for  a  considerable  time.  While 
this  procedure  yielded  a  milk  of  very  excellent  keeping  qualities,  it 
was  soon  found  to  have  its  disadvantages  in  that  it  developed  certain 
features  disagreeable  to  the  taste,  which  after  a  time  made  it  decidedly 
distasteful  and  even  repulsive  to  some  people;  but  still  more  important 
were  the  facts  that  it  became  less  easily  digestible  and  assimilable, 
and  that  it,  when  fed  exclusively  to  infants  and  very  young  children, 
produced  rickets  and  scurvy,  with  anemia  and  other  metabolic  and 
developmental  disturbances.  The  use  of  fully  sterilized  milk  has  today 
been  almost  entirely  abandoned  in  the  feeding  of  infants,  children, 
convalescents,  or  invalids,  and  instead  pasteurized  milk  is  advocated 
by  many. 


506  THE  PASTEURIZATION  OF  MILK 

The  terms  sterilization  and  pasteurization  with  reference  to  milk 
are  today,  unfortunately,  still  used  somewhat  indiscriminately,  and, 
in  fact,  no  strictly  scientific  definition  of  the  term  pasteurization  is  in 
existence.  Tjaden,  in  the  chapter  on  "Sterilization  and  Pasteur- 
ization" in  Sommerfeld's  Manual  on  Milk,  define  pasteurization 
as  the  heating  of  milk  up  to  98°  to  99°  C.;  sterilization  as  the  heating 
to  the  actual  boiling  temperature  or  beyond  it;  and  Foersterization,  as 
the  heating  at  60°  C.  for  one  hour.  Rosenau  defines  pasteurization 
as  applied  to  milk  in  heating  it  to  60°  C.  for  twenty  minutes,  followed 
by  rapid  cooling. 

As  the  object  of  pasteurization,  Tjaden  designates : 

1.  A  better  and  more  complete  separation  of  the  milk  into  its  com- 
ponent constituents. 

2.  To  make  milk  products  more  tasty  and  to  impart  to  them  better 
keeping  qualities. 

3.  To  improve  the  keeping  quality  of  the  milk  as  a  whole. 

4.  To  destroy  disease  germs  which  may  possibly  be  present  in  milk. 
It  has  been  ascertained  that  the  yield  in  butter  fat  in  milk  treated 

in  the  centrifuge  is  much  better  at  45°  to  80°  C.  than  at  lower  temper- 
atures. Milk  products  derived  from  heated  milk  and  treated  with 
pure  cultures  of  certain  fermentative  bacteria,  permitted  to  act  upon 
cream  and  casein  in  the  manufacture  of  butter  and  cheese,  were  also 
found  to  have  a  much  finer  taste  and  flavor  than  the  same  edibles 
prepared  from  raw  milk  in  which  other  bacteria  present  modify  the 
special  fermentations.  While  pasteurization  improves  the  keeping 
qualities  of  milk  this  is  only  true  as  long  as  the  milk  after  heating  is 
cooled  rapidly,  kept  cool,  and  not  dispensed  in  dirty  vessels  which 
would  again  lead  to  contamination  and  the  rapid  multiplication  of 
bacteria.  Pasteurized  milk  has  lost  the  power  to  inhibit  the  growth 
of  bacteria  for  some  time,  and  those  bacteria -which  do  multiply  in 
it  are  not  the  harmless  lactic-acid  bacteria  but  the  spore-forming, 
peptonizing,  putrefactive  bacteria. 

The  pasteurizers  used  in  the  heating  of  the  milk  on  a  large  com- 
mercial scale  are  generally  of  either  one  of  two  types.  The  milk 
which  goes  rapidly  through  an  apparatus  in  a  continuous  stream,  is 
either  heated  for  a  very  short  time  to  a  comparatively  high  temper- 
ature or  it  remains  in  the  apparatus  for  a  comparatively  long  time,  is 
agitated  during  this  period,  and  is  kept  at  a  proportionately  lower 
temperature.  This  is  called  the  discontinuous  method. 

The  object  is  always  the  destruction  of  most  bacteria  of  any  kind, 
and  the  destruction  of  all  pathogenic  bacteria.  The  most  important 
of  the  latter  is  the  tubercle  bacillus.  Numerous  scientific  investiga- 
tions have  dealt  with  temperatures  and  periods  necessary  to  destroy 
it  in  milk.  The  results  have  furnished  by  no  means  uniform  data,  but 
it  appears  to  be  generally  conceded  (Tjaden,  Weigmann,  and  others) 
that  in  the  continuous  method  heating  to  85°  C.  (185°  F.)  for  one  to 
two  minutes  is  generally  sufficient  to  kill  tubercle  bacilli.  Tjaden, 


THE  PASTEURIZATION  OF  MILK  507 

Koske,  and  Hertel,  however,  have  made  a  series  of  experiments  with 
milk  from  cows  with  udder  and  other  forms  of  tuberculosis  and  have 
subjected  the  milk  to  temperatures  of  85°,  90°,  95°,  and  100°  C.  by 
the  continuous  and  discontinuous  method  in  apparatuses  of  various 
construction.  Some  of  the  tests  furnished  the  very  remarkable  result 
that  at  any  of  the  above  temperatures  tubercle  bacilli  in  the  milk 
from  cows  with  udder  tuberculosis  were  not  destroyed  and  were  able  to 
produce  tuberculosis  by  subsequent  inoculations  into  guinea-pigs  and 
in  feeding  to  young  pigs.  Such  positive  results  were  obtained  in  four 
series  of  experiments  in  which  the  continuous  method  and  100°  C* 
were  used,  and  in  several  experiments  in  which  the  discontinuous 
method,  temperatures  of  98°  C.,  and  time  exposures  from  sixty  to 
one  hundred  and  five  seconds  were  employed.  The  milk  samples 
used  in  these  tests  were  very  bad  and  some  of  the  resisting  tubercle 
bacilli  were  evidently  inclosed  in  casein  or  pus  coagula.  Yet  it 
cannot  be  denied  that  under  certain  conditions  tubercle  bacilli  in 
milk  will  survive  exposures  to  100°  C.  and  to  98°  C.  for  over  one 
hundred  seconds.  Foster  and  DeMan,  using  milk  from  tubercular 
udders,  found  that  tubercle  bacilli  were  killed  at  the  following  tem- 
peratures and  periods : 

At  55°,C.  (131°  F.)  after  four  hours. 

At*60°tC.  (140°  F.)  after  one  hour. 

At  65°  C.  (149°  F.)  after  fifteen  minutes. 

At  70°  C.  (158°  F.)  after  ten  minutes. 

At  80°  C.  (176°  F.)  after  five  minutes. 

At  90°  C.  (194°  F.)  after  two  minutes. 

At  95°  C.  (203°  F.)  after  one  minute. 

That  commercial  pasteurization,  as  frequently  practised  in  this 
country,  does  not  kill  all  tubercle  bacilli  in  milk  has  been  proved  a 
number  of  times  by  guinea-pig  inoculation  of  pasteurized  milk. 

The  heating  of  the  milk  in  pasteurization  produces  certain  changes. 
When  milk  is  heated  by  the  continuous  method  to  85°  C.,  but  cooled 
rapidly,  little  change  in  taste  and  smell  is  produced,  though  a  trace  of 
"boiled  taste"  may  be  noticeable.  This  is,  as  a  rule,  more  marked  in  the 
longer  heating  at  lower  temperatures.  The  same  is  true  of  cream  and 
skimmed  milk  after  pasteurization.  The  formation  of  cream  occurs 
somewhat  more  slowly  in  heated  than  in  non-heated  milk,  but  the 
yield  in  butter  fat  is  somewhat  larger  in  the  former,  as  already  stated. 
The  greatest  effect  of  the  heating  of  the  milk  in  pasteurization  is 
undoubtedly  exerted  upon  the  enzymes  of  the  milk.  Even  if  they 
are  not  all  completely  destroyed  their  action  is  undoubtedly  much 
weakened  and  modified  so  that  from  a  purely  physiological  standpoint 
pasteurized  milk  is  of  inferior  value  in  the  nutrition  of  infants 
when  compared  with  first-class  raw  milk.  Tests  have  been  devised 
particularly  for  one  of  the  groups  of  enzymes  in  milk,  i.  e.,  the  per- 
oxydases  which  are  oxidizing  ferments  and  transfer  the  oxygen  in 
metabolic  processes  of  the  organism.  These  tests  can  be  used  to 
discover  whether  milk  has  been  heated  to  a  certain  temperature. 


508  THE  PASTEURIZATION  OF  MILK 

Starch's  Test  for  Enzymes  in  Milk. — This  test  is  generally  employed 
in  testing  milk  for  the  presence  of  the  peroxydases.  It  is  made  as 
follows :  Take  10  c.c.  of  milk  in  a  test-tube,  add  one  or  two  drops  of 
dilute  hydrogen  peroxide  (H2O2),  and  mix  well  by  shaking,  then  add 
two  drops  of  a  2  per  cent,  solution  of  paraphenylendiamine  and 
shake  again.  In  raw  milk  an  indigo-blue  color  is  produced,  depending 
upon  the  presence  of  active  oxydases.  The  test  may  also  be  made, 
according  to  Rullman,  as  a  contact  ring  test  as  follows:  Take  10  c.c. 
of  milk  in  a  test  tube,  add  10  drops  of  a  3  per  cent,  solution  of  hydrogen 
peroxide,  and  shake  well.  Hold  test-tube  very  obliquely  and  add 
slowly  from  a  pipette  1  c.c.  of  a  2  per  cent,  solution  of  paraphenylen- 
diamine, allowing  it  to  flow  along  the  glass  on  the  surface  of  the  milk. 
A  grayish-blue  ring  is  formed  at  the  zone  of  contact  between  the  milk 
and  the  test  fluid.  According  to  Kastle,  the  reaction  is  delayed  in 
milk  which  has  been  heated  to  70°  C.  for  fifteen  minutes,  while  it  is 
absent  after  heating  to  70°  C.  for  one  hour.  It  is  somewhat  delayed 
after  heating  to  60°  C.  for  one  hour,  but  not  affected  >  after  thirty 
minutes'  heating  to  60°  C. 

Home  Pasteurization. — Directions  for  the  home  pasteurization  of 
milk  as  given  by  Rogers  in  Circular  No  152,  of  the  Bureau  of  Animal 
Industry,  are  as  follows: 

"  Milk  is  most  conveniently  pasteurized  in  the  bottles  in  which  it  is 
delivered.  To  do  this  use  a  small  pail  with  a  perforated  false  bottom. 
An  inverted  pie  tin  with  a  few  holes  punched  in  it  will  answer  the 
purpose.  This  will  raise  the  bottles  from  the  bottom  of  the  pail, 
thus  allowing  a  free  circulation  of  water  and  preventing  bumping  of 
the  bottles.  Punch  a  hole  through  the  cap  of  one  of  the  bottles  and 
insert  a  thermometer.  The  ordinary  floating  type  of  thermometer 
is  likely  to  be  inaccurate  and  if  possible  a  good  thermometer  with 
the  scale  etched  on  the  glass  should  be  used.  Set  the  bottles  of  milk 
in  the  pail  and  fill  the  pail  with  water  nearly  to  the  level  of  the  milk. 
Put  the  pail  on  the  stove  or  over  a  gas  flame  and  heat  it  until  the 
thermometer  in  the  milk  shows  not  less  than  150°  F.,  nor  more  than 
155°  F.  The  bottles  should  then  be  removed  from  the  water  and 
allowed  to  stand  from  twenty  to  thirty  minutes.  The  temperature 
will  fall  slowly,  but  may  be  held  more  uniformly  by  covering  the  bottles 
with  a  towel.  The  punctured  cap  should  be  replaced  by  a  new  one, 
or  the  bottle  should  be  covered  with  an  inverted  cup. 

"After  the  milk  has  been  held  as  directed  it  should  be  cooled  as 
quickly  and  as  much  as  possible  by  setting  in  water.  To  avoid  danger 
of  breaking  the  bottle  by  too  sudden  change  of  temperature  this 
water  should  be  warm  at  first.  Replace  the  warm  water  slowly  with 
cold  water.  After  cooling,  milk  should  in  all  cases  be  held  at  the 
lowest  available  temperature. 

"  This  method  may  be  employed  to  retard  the  souring  of  milk  or 
cream  for  ordinary  use.  It  should  be  remembered,  however,  that 
pasteurization  does  not  destroy  all  bacteria  in  milk,  and  after  pasteur- 


ADVANTAGES  AND  DISADVANTAGES  OF  PASTEURIZATION     509 

ization  it  should  be  kept  cold  and  used  as  soon  as  possible.  Cream 
does  not  rise  as  rapidly  or  separate  as  completely  in  pasteurized  milk 
as  in  raw  milk." 

Advantages  and  Disadvantages  of  Pasteurization. — Pasteurization  has 
its  advantages  and  its  disadvantages.  It  has  its  enthusiastic  advo- 
cates and  those  who  are  opposed  to  the  wholesale  pasteurization  of 
the  milk  supply  of  a  big  city.  Rosenau  gives  his  views  as  follows : 

"  One  of  the  chief  objections  to  pasteurization  is  that  it  promotes 
carelessness  and  discourages  the  efforts  to  produce  clean  milk.  It 
is  believed  that  the  general  adoption  of  pasteurization  will  set  back 
improvements  at  the  source  of  supply  and  encourage  dirty  habits. 
It  will  cause  the  farmers  and  those  who  handle  the  milk  to  believe 
that  it  is  unnecessary  to  be  quite  so  particular,  as  the  dirt  that  gets 
into  the  milk  is  going  to  be  cooked  and  made  harmless.  It  is  not 
proposed  that  pasteurization  shall  take  the  place  of  inspection  and 
improvements  in  dairy  methods.  To  insure  the  public  a  pure  and 
safe  milk  supply  should  be  regarded  as  one  of  the  most  important 
duties  of  the  health  officer.  Whether  pasteurization  is  adopted  by 
a  city  for  its  general  milk  supply  or  not,  no  milk  should  be  accepted 
that  does  not  comply  with  certain  reasonable  chemical  and  bac- 
teriologic  standards.  This  would  aid  the  inspectors  in  enforcing 
good  dairy  methods.  Pasteurization  then  must  not  be  used  as  an 
excuse  to  bolster  up  milk  unfit  for  home  consumption.  To  insure 
this  end  the  health  officer  should  have  authority  to  condemn  and 
destroy  bad  milk,  whether  or  not  pasteurization  is  practised. 

"There  is  a  prevalent  impression  that  the  pasteurization  of  milk 
improves  that  important  article  of  diet.  Heating  does  not  render 
milk  better  in  any  way  as  a  food.  All  it  does  is  to  destroy  certain 
bacteria  and  some  of  their  toxic  products.  It  checks  certain  pro- 
cesses of  fermentation  and  putrefaction,  thus  rendering  the  milk 
safer.  On  the  other  hand  the  evidence  seems  clear  that  the  pasteur- 
ization of  milk  at  60°  C.  for  twenty  minutes  does  not  appreciably 
deteriorate  its  quality  or  lessen  its  food  value. 

"Theoretically,  pasteurization  should  not  be  necessary;  practically, 
we  find  it  forced  upon  us.  The  heating  of  milk  has  certain  dis- 
advantages which  must  be  given  consideration,  but  it  effectually 
prevents  much  disease  and  death,  especially  in  infants  during  the 
summer  months." 

Among  the  objections  to  pasteurization,  Jensen  mentioned  the 
following:  "Even  by  the  use  of  a  self-regulating  pasteurizer  it  is 
difficult  to  provide  absolute  guarantee  that  all  milk  has  been  heated 
to  the  required  temperature.  To  a  certain  degree  pasteurization 
may  conceal  a  tainted  condition,  which  exists  before  heating.  Quite 
an  abundance  of  bacteria  of  putrefaction  and  other  bacteria  may 
be  present  or  the  lactic-acid  fermentation  may  have  begun  to  take 
place;  these  bacteria  are  killed  by  pasteurization;  consequently  the 
fermentation  and  changes  that  were  under  way  are  interrupted. 


510  THE  PASTEURIZATION  OF  MILK 

Under  such  circumstances  one  cannot  tell  by  the  appearance  or 
taste  of  the  milk  that  it  is  damaged  and  that  it  contains  the  product 
of  decompositon  of  the  albumin,  or  possibly  even  toxic  substances. 
On  the  whole  there  is  no  way,  at  the  present  time,  of  determining 
whether  or  not  pasteurized  milk  was  damaged  before  it  was  heated, 
while  with  respect  to  raw  milk  the  keeping  quality  and  bacterial 
content  furnish  sufficient  evidence  regarding  its  true  condition.  The 
bacteria  surviving  pasteurization  are,  for  the  most  part,  the  quick- 
growing  bacteria  of  putrefaction  which  are  inhibited  in  raw  milk 
by  the  lactic-acid  bacteria,  but  in  pasteurized  milk  they  multiply  very 
fast  and  undoubtedly  they  are  capable  of  generating  poisonous 
substances.  It  has  been  suggested,  therefore,  that  a  pure  culture  of 
lactic-acid  bacteria  be  added  to  milk  after  pasteurization  in  order  to 
check  the  bacteria  of  putrefaction.  In  purchasing  pasteurized  milk 
one  cannot  tell  if  it  be  fresh  or  old  and  cannot  determine  from  its 
appearance  whether  putrefaction  has  begun  or  if  only  a  few  bacteria 
are  present.  If  we  compare  the  advantages  and  disadvantages  it  will 
be  found  that  there  is  serious  doubt  as  to  whether  it  is  advisable  to 
endeavor  to  obtain  general  pasteurizaton  of  market  milk,  as  has  been 
suggested  in  Germany." 

While  there  can  be  no  objection  to  the  home  pasteurization  of 
milk,  in  order  to  destroy  pathogenic  bacteria  which  might  be  present, 
and  to  impart  to  milk  better  keeping  qualities,  compulsory  pasteur- 
ization of  most  of  the  market  milk  supply,  decreed  by  city  ordinances 
has  appeared  very  objectionable  to  the  author,  and  he  has  had  occasion 
to  express  his  views  on  such  measures  as  they  have  been  enacted  in 
the  city  of  Chicago. 

Summary  of  Objections. — A  summary  of  these  objections  against 
the  wholesale  pasteurization  of  the  milk  supply  of  a  big  city  is  con- 
tained in  the  following  paragraphs: 

1.  It  is  known  that  the  exclusive  feeding  of  sterilized  or  pasteur- 
ized milk  to  infants  and  children  has  a  tendency  to  produce  rickets 
and  scurvy.  This  is  due  to  the  fact  that  any  effort  at  pasteurization 
which  will  destroy  a  high  percentage  of  bacteria  will  also  destroy 
several  very  important  soluble  ferments  or  enzymes  contained  in 
milk.  The  latter  are  absolutely  necessary  for  the  proper  nutrition 
of  the  infant  body,  which  does  not  yet  furnish  these  ferments,  as  is 
done  in  later  life.  The  production  of  rickets  has  been  frequently 
observed  not  only  in  man  but  as  well  in  some  of  the  lower  animals, 
particularly  in  small,  fancy,  high-bred  dogs,  which  often  have  to  be 
brought  up  on  pasteurized  milk  because  the  mother's  milk  is  secreted 
in  insufficient  quantity  and  the  puppies  do  not  well  tolerate  raw 
cow's  milk.  On  the  other  hand  it  is  claimed  that  the  feeding  of 
pasteurized  cow's  milk  to  calves  has  not  been  followed  by  any  evil 
consequences  to  the  cattle  stock  of  Denmark,  where  this  method  of 
feeding  has  been  practised  quite  extensively  for  a  number  of  years. 
However,  observations  made  on  calves  fed  with  pasteurized  cow's 


QUESTIONS 

milk  cannot  be  made  applicable  to  human  infants  fed  on  the  same 
article  of  diet. 

2.  Pasteurization    of    milk    makes    a    subsequent    bacteriological 
examination  and  estimation  of  milk,  as  it  originally  was,  impossible. 
The  evidence  of  the  already  undesirable  and  spoiled  character  of  the 
milk  will  be  destroyed  by  pasteurization. 

3.  Pasteurization  of  milk  practically  destroys  the  so-called  lactic- 
acid  bacteria;  hence,  pasteurized  milk  will  not  easily  turn  sour,  but  it 
will  undergo  putrefactive  changes,  which,  while  not  readily  apparent  to 
the  senses  of  taste  and  smell,  make  it  a  very  improper  and  dangerous 
article  of  diet  for  the  feeding  of  infants  and  children. 

4.  It  appears  impossible  to  control,  at  all  times,  the  whole  supply 
of  pasteurized  milk  of  a  large  city;  hence,  there  is  great  danger  that 
much  improperly  pasteurized  milk  may  be  passed  through  its  market. 

5.  Pasteurization  does  not  attack  the  evil  of  milk  from  tubercular 
and  otherwise  diseased  cows  at  its  root,  but  can  at  best  be  looked 
upon  as  a  makeshift  to  lessen  the  dangers  of  a  milk  which  should 
from  the  beginning  have  been  condemned  as  an  improper  food  for 
infants  and  young  children. 

6.  Pasteurization  is  chiefly  directed  against  the  dangers  of  spread- 
ing tuberculosis  from  the  cow  to  the  infant  and  the  child.     It  has 
been  frequently  shown  that  ordinary  commercial  pasteurization,  as 
practised  in  some  of  the  cities  of  this  country,  does  not  safely  kill 
the  tuberculosis  germ,  and  milk  which  has  been  sold  as  pasteurized, 
for  instance,  in  the  city  of  New  York,  has  effectively  infected  experi- 
mental animals  with  tuberculosis. 

7.  It  has  been  noted   in  Rochester,  N.  Y.,  and  in  other  cities, 
that  compulsory  pasteurization  has  caused  great  deterioration  in  the 
character  of  the  milk  supply  furnished  to  the  markets  where   such 
laws  were  in  force. 

8.  The  only  proper  measure   to   improve   the   character  of   the 
milk  supply  and  to  safeguard  the  people  against  the  dangers  which 
lurk  in  tuberculosis  and  otherwise  infected  milk  is  to  tuberculin- 
test  milch  cows;  to  inspect  dairies  thoroughly,  and  to  enforce  rules, 
requiring  dairymen  to  keep  only  healthy  cows  under  proper  hygienic 
conditions  and  under  constant  veterinary  supervision. 


QUESTIONS 

1.  Do  the  milk-ducts  of  the  udders  of  healthy  cows  contain  any  bacteria? 
Discuss  the  bacterial  content  of  the  fore-milk. 

2.  What  is  the  first  effect  of  raw  milk  upon  the  bacterial  content?    What  is 
this  effect  due  to  ? 

3.  What  is  the  effect  upon  the  bacterial  count  of  keeping  milk  under  lower 
id  higher  temperatures? 

4.  Discuss  the  significance  of  the  number  of  bacteria  in  milk  according  to 
the  views  of  various  authors. 

5.  Describe  in  detail  the  method  of  estimating  the  number  of  bacteria  per 
c.c.  of  milk. 


512  THE  PASTEURIZATION  OF  MILK 

6.  Does  this  method  furnish  absolutely  accurate  values?    If  not,  why  not? 

7.  Describe  in  detail  the  method  of  estimating  the  acidity  of  cow's  milk. 

8.  How  are  the  results  of  bacterial  counts  rounded  off  when  recording  the 
results  of  bacterial  milk  examinations  ? 

9.  How  are  bacterial  counts  of  certified  milk  expressed  ? 

10.  What  is  certified  milk? 

11.  Give  in  outline  the  rules  under  which  certified  milk  should  be  produced. 

12.  What  are  the  duties  of  the  veterinarian  in  connection  with  the  production 
of  certified  milk? 

13.  Where  does  the  term  pasteurization  come  from? 

14.  Give  a  definition  of  it. 

15.  What  is  the  difference  between  sterilization  and  pasteurization? 

16.  What  is  the  difference  between  continuous  and  discontinuous  pasteuri- 
zation? 

17.  What  are  the  four  objects  of  the  pasteurization  of  milk? 

18.  What  exposures  to  heat  are  necessary  to  kill  tubercle  bacilli  in  milk? 

19.  Under  what  conditions  are  tubercle  bacilli  in  milk  particularly  difficult  to 
destroy  ? 

20.  Has  commercial  pasteurization  in  the  past  been  generally  a  guarantee  for 
the  destruction  of  the  tubercle  bacilli  in  milk  ? 

21.  What  is  the  effect  of  a  reliable  pasteurization  upon  the  enzymes  in  milk  ? 
22^  What  are  enzymes? 

23.  Describe  Storch's  test  for  detecting  peroxydases  in  milk? 

24.  Describe  Rullman's  modification  of  the  Storch  test. 

25.  What  is  the  effect  upon  peroxydases  in  milk  if  the  latter  is  heated  for  one 
hour  at  70°  C.? 

26.  Describe  the  method  of  home  pasteurization  of  milk. 

27.  Discuss  the  advantages  and  disadvantages  of  pasteurization. 


CHAPTEE    XLVIII. 

BACTERIA  IN  BUTTER  AND  CHEESE-MAKING. 

THE  most  important  products  prepared  from  milk,  which  is  not 
consumed  as  such,  are  butter  and  cheese.  The  former  may  be 
obtained  from  sweet  milk,  sour  milk,  sweet  cream,  or  sour  cream. 
Cream  may  be  permitted  to  separate  spontaneously  from  milk,  but 
this  is  rarely  done  today,  when  most  cream  is  obtained  by  centrifuging 
the  milk  in  a  centrifuging  apparatus  or  cream  separator.  This  has 
the  advantage  that  the  separation  occurs  more  rapidly  and  is  more 
complete;  in  fact,  apparatuses  have  been  constructed  which  will 
leave  in  the  skimmed  milk  only  0.06  to  0.12  per  cent,  butter  fat  and 
will  remove  from  the  whole  milk  with  the  cream  over  96  per  cent,  of 
the  butter  fat.  Butter  is  usually  prepared  from  sour  cream.  The 
souring  of  the  cream  may  have  been  permitted  to  occur  spontaneously, 
generally  within  thirty  to  thirty-six  hours,  by  the  action  of  lactic  acid 
bacteria,  or  it  may  have  been  brought  about  by  the  addition  of  a 
so-called  "starter,"  or  "Saurewecker"  (German).  This  starter  con- 
sists of  whole  milk  or  skimmed  milk  which  has  become  sour  spon- 
taneously or  a  culture  of  lactic-acid  bacteria.  The  employment  of 
the  artificially  prepared  starters  owes  its  origin  to  the  investigations  of 
Storch,  Weigmann,  and  Conn.  Storch,  in  Denmark,  and  Weigmann, 
in  Germany,  have  shown  that  the  souring  of  the  cream  was  due  to 
lactic-acid  bacteria,  and  Leichmann  had  demonstrated  that  it  was  par- 
ticularly due  to  the  Streptococcus  lacticus.  Cultures  of  this  organism, 
also  containing  some  other  bacteria  and  yeast  cells  which  have  the 
power  to  produce  an  agreeable  aroma  in  the  butter,  have  been  used 
more  and  more  during  the  last  twenty  years  in  souring  the  cream  for 
the  preparation  of  butter.  It  was  found,  however,  that  the  aroma 

I  microorganisms  must  be  used  very  carefully,  since  some  of  them, 
particularly  under  certain  conditions,  have  a  tendency  to  split  some 
af  the  butter  fat,  forming  butyric  acid  and  making  the  butter  rancid, 
[t  has  also  become  a  practice  frequently  to  pasteurize  the  milk  or 
cream  which  is  subsequently  soured  by  the  starter.  Aside  from 
hygienic  considerations  the  advantage  of  this  is  that  the  souring  is 
produced  by  definite  organisms  and  not  by  a  variety  contained 
originally  in  the  milk,  which  might  impart  undesirable  flavors  or 
other  objectionable  properties  to  the  butter.  The  sour  cream  is 
subsequently  churned  in  apparatuses  of  various  types,  in  which  it  is 
subjected  to  violent  agitation  and  in  consequence  the  globules  of 
butter  fat  become  confluent  and  most  of  the  watery  part  of  the  cream 
33 


514  BACTERIA  IN  BUTTER  AND  CHEESE-MAKING 

becomes  separated  as  buttermilk.  The  latter  generally  has  the 
following  composition :  Water,  over  90  per  cent. ;  fat,  about  J  per  cent. ; 
nitrogenous  compounds,  3.4  per  cent.;  lactose,  4.7  per  cent.;  ash, 
0.7  per  cent. 

The  artificial  starters  used  in  the  souring  of  cream  as  employed 
in  this  country  are  either  in  the  form  of  a  powder  or  in  the  form  of 
a  liquid  culture.  They  are  first  increased  before  being  added  to  the 
cream,  a  procedure  called  the  "building  up  of  the  starter."  This  is 
done  by  adding  a  freshly  opened  package  or  bottle  of  the  starter  to  a 
quart  of  skim  milk,  whole  milk,  or  cream  which  has  been  sterilized  or 
pasteurized  and  cooled  down  to  60°  F.,  and  stirring  and  mixing  it 
thoroughly  with  the  milk.  The  latter  is  then  kept  at  65°  F.,  protected 
against  dirt  and  other  contamination.  When  quite  sour,  but  before 
coagulation  has  occurred,  the  increased  or  built-up  starter  is  added 
to  the  cream  which  is  to  be  soured  or  ripened.  If  the  cream  used 
has  been  pasteurized  or  has  come  from  pasteurized  milk,  more  of  the 
starter  is  needed  than  if  this  is  not  the  case,  and  thus,  according  to 
varying  circumstances  from  4  to  10  per  cent,  of  the  starter  are  added. 
The  use  of  pasteurized  cream  for  butter  making  is  very  prevalent 
in  some  European  countries,  because  the  butter  so  obtained,  owing 
to  the  non-development  of  certain  undesirable  bacteria,  is  more 
uniform  in  character  and  less  liable  to  show  objectionable  features. 
In  the  United  States  artificially  prepared  starters  are  often  used  on 
unpasteurized  cream.  In  such  cases  the  bacteria  of  the  starter  act  in 
combination  with  the  bacteria  already  present  in  the  cream  and  the 
results  are  not  as  satisfactory  as  when  pasteurized  cream  is  used. 

Conn  (Agricultural  Bacteriology},  in  discussing  the  use  of  starters 
in  our  country,  says :  "The  fact  that  starters,  with  or  without  pasteur- 
ization have  become  almost  universally  used  among  the  better  class 
of  creameries  is  in  itself  sufficient  proof  that  they  are  of  practical 
value.  Their  advantage  lies  in  four  directions : 

"1.  They  enable  the  buttermaker  to  handle  his  cream  more  easily 
and  uniformly.  He  can  regulate  the  ripening  in  such  a  way  that 
his  cream  will  always  be  of  a  certain  grade  of  ripeness  at  a  certain 
time  of  the  day;  for  a  little  experience  tells  him  how  much  of  his 
culture,  under  proper  conditions,  should  be  added  to  the  cream  to 
produce  the  proper  grade  of  ripening  at  the  particular  time  when  he 
desires  to  churn. 

"2  The  use  of  starters  has  produced  a  greater  uniformity  in  the 
grade  of  butter.  The  buttermaker  can  depend  more  certainly 
upon  producing  butter  of  a  high  grade,  month  after  month,  than  he 
can  without  the  starter.  There  is  a  general  belief  also  among  those 
who  have  tested  the  butter  in  countries  where  starters  are  widely 
used  that  there  is  an  improvement  in  the  average  quality  of  the 
butter  as  well  as  in  its  uniformity. 

"3.  It  has  become  pretty  definitely  agreed  that  the  flavor  of  butter 
is  improved  by  the  use  of  such  cultures.  It  is  somewhat  difficult 


BACTERIA  IN  CREAM  AND  BUTTER  515 

to  obtain  definite  proof  of  this,  owing  to  the  uncertainty  of  scores 
in  butter  tests.  But  the  fact  that  all  good  dairies  use  them  is  sufficient 
testimony  to  their  value  in  improving  the  general  quality  of  the 
butter. 

"4.  They  are  the  best  means  of  remedying  butter  faults.  Every 
creamery  has  experiences  of  deterioration  in  the  flavor  of  the  butter 
without  any  visible  cause.  Such  troubles  are  known  to  be  due  com- 
monly to  the  growth  of  unusual  and  undesirable  bacteria  in  the  cream. 
When  they  are  discovered  the  sterilizing  of  the  dairy  utensils  and 
the  use  of  a  larger  quantity  of  vigorous  starter  will  generally  remedy 
the  trouble  at  once.  Moreover,  the  constant  use  of  a  starter  goes  a 
long  way  toward  preventing  these  ''faults."  It  is  doubtful  whether  the 
use  of  starters  produces  a  butter  of  a  character  superior  to  the  best 
butter  made  without  them.  Indeed,  some  think  that  it  is  not  quite 
^qual  to  the  best  butter  made  without  starters.  But  the  uniformly 
high  grade  of  culture  butter  is  admitted,  and  the  greater  satisfaction 
in  being  able  to  control  the  process  has  caused  the  wide  adoption  of 
starters  among  buttermakers." 

Bacteria  in  Cream  and  Butter. — The  ripening  of  the  cream  is  due  to 
bacterial  growth,  chiefly  lactic-acid  bacteria,  with  the  formation  of 
lactic  acid  from  lactose.  The  proper  ripening  of  the  cream,  however, 
cannot  be  brought  about  by  simply  adding  to  it  a  certain  amount  of 
lactic  acid.  This  shows  that  a  variety  of  bacterial  enzymes  are 
active  in  ripening  cream  so  that  it  will  be  in  the  best  shape  for  butter- 
making;  this  is  generally  the  case  when  the  acidity  present  is  equal 
to  0.5  to  0.65  per  cent.  An  enormous  increase  of  bacteria  occurs  in 
cream  during  the  changes  which  lead  to  ripening.  From  2,000,000  to 
3,000,000  bacteria  per  c.c.  may  have  been  present  in  the  sweet  cream, 
and  when  it  is  ready  or  ripe  for  butter-making  the  number  may  have 
increased  to  several  hundred  million  per  c.c.  and  even  to  2,000,000,000. 
The  ripening  is  best  allowed  to  continue  at  65°  F.,  because  at  that 
temperature  the  danger  of  development  of  undesirable  butter-spoiling 
bacteria  is  much  less  than  at  higher  temperatures.  The  growth  of 
bacteria  in  ripening  cream  is  generally  stopped  by  churning ;  many  of 
them  are  removed  with  the  buttermilk  and  more  with  the  subsequent 
washing  and  kneading  of  the  butter.  While  butter  is  being  kept 
the  number  of  bacteria  rapidly  decreases,  particularly  in  salted  butter. 
Conn  gives  the  following  examples :  Number  of  bacteria  present  per 
gram  of  butter  two  hours  old,  54,000,000;  one  day  old,  26,000,000; 
four  days  old,  2,000,000;  thirty  days  old,  300,000.  There  are,  however, 
some  bacteria  present  even  in  very  old  butter.  In  order  to  protect 
butter  against  subsequent  changes  it  must  be  kept  at  a  low  tem- 
perature and  protected  against  light  and  air.  If  this  is  not  done  it 
will  soon  become  rancid,  i.  e.,  some  of  the  butter  fat  is  decomposed 
and  changed  into  butyric  acid,  a  fermentative  process  due  to  certain 
bacteria  and  their  special  enzyme. 


516  BACTERIA  IN  BUTTER  AND  CHEESE-MAKING 

Bacteria  and  Other  Microorganisms  in  the  Ripening  of  Cheese.— 
Proteids  in  Milk. — Cow's  milk  contains  about  3  to  4  per  cent,  of 
nitrogenous  organic  compounds  or  proteids,  and  these  are,  as  shown 
by  Hammarsten,  not  of  one  kind,  but  three  chemically  different  bodies 
known  as  casein,  lactalbumin,  and  globulin.  The  casein  is  equal  to 
about  80  per  cent,  of  the  entire  amount  of  proteids,  and  it  is  present 
in  milk  as  a  calcium  compound.  It  is  not  in  true  solution,  but  in  a 
swollen,  finely  divided  condition  known  as  the  colloidal  state.  When 
milk  is  acidulated  beyond  a  certain  degree,  either  by  the  addition  of 
acid  from  without  or  by  the  growth  and  development  of  lactic-acid 
bacteria,  the  casein  is  precipitated  as  a  more  or  less  finely  flocculent 
mass.  It  is  more  finely  flocculent  in  human  milk,  more  coarsely 
flocculent  in  cow's  milk.  When  this  change  has  occurred  the  milk 
is  said  to  have  coagulated. 

Coagulation  of  Milk. — Coagulation  of  milk  can  be  brought  about 
by  another  procedure  aside  from  acidulation,  namely,  by  the  addition 
of  an  enzyme  called  labferment,  or  rennet.  This  enzyme  is  furnished 
by  the  mucous  membrane  of  the  stomach  of  animals,  and  also  by  a 
number  of  bacteria,  as  first  shown  by  Ducleaux,  who  demonstrated 
that  certain  bacteria  growing  in  milk  coagulate  it  by  the  aid  of  this 
ferment.  Conn  succeeded  in  separating  the  rennet  enzyme  from 
the  bacteria  which  had  produced  it.  While  very  similar  in  action  the 
bacterial  rennet  is  not  absolutely  identical  with  the  rennet  obtained 
from  calves'  stomachs  or  the  stomachs  of  other  mammals.  The 
bacterial  rennet  can  also  coagulate  sterilized  milk,  the  latter  only 
raw  milk.  There  are  quite  a  number  of  bacteria  which  can  coagulate 
milk  by  the  aid  of  their  rennet  enzyme  even  without  acidulating  it. 
Among  such  bacteria  are  particularly  the  potato  bacilli  (Bacillus 
mesentericus  vulgatus  and  other  bacteria  of  this  group).  The  rennet 
prepared  from  calves'  stomachs  cannot  coagulate  boiled  milk  because  it 
acts  only  in  the  presence  of  soluble  lime  salts,  and  these  are  precipitated 
when  milk  is  boiled;  it  can  likewise  not  act  in  an  alkaline  solution, 
but  acts  best  in  a  slightly  acid  medium  at  a  temperature  of  37°  C. 
At  25°  C.  the  action  is  slow;  at  45°  C.  it  does  not  take  place,  and  at 
70°  C.  the  enzyme  is  destroyed. 

The  Formation  of  the  Curd. — In  order  to  prepare  cheese,  milk  may 
be  coagulated  by  the  addition  of  the  lab-  or  rennet  enzyme1  from 
calves'  stomachs,  by  permitting  it  to  become  sour  spontaneously,  or 
by  adding  milk  already  soured  or  a  starter.  According  to  the  method 
used  in  coagulation,  cheeses  are  distinguished  as  rennet  milk  cheeses 
and  sour  milk  cheeses.  The  soft,  spongy  mass,  full  of  fluid,  which  is 
formed  when  milk  has  been  coagulated  by  either  one  of  the  two 
methods,  is  called  a  curd.  Most  of  the  whey  may  be  pressed  out  of 
the  curd  or  a  considerable  portion  may  be  left  in  it.  In  the  former 
case  the  comparatively  dry  raw  material  will  form  the  hard,  in  the  latter 

1  An  exceedingly  small  amount  of  rennet  will  coagulate  a  very  large  amount  of  milk. 


BACTERIA  AND  OTHER  MICROORGANISMS  IN  CHEESE      517 

case  the  soft  cheese.  The  raw  material  so  obtained  is  still  simply  curd 
and  becomes  cheese  only  after  going  through  a  process  of  ripening 
with  various  changes  depending  upon  the  activity  of  bacteria  and  other 
microorganisms.  In  addition  to  the  proteids,  the  curd  contains  a 
variable  amount  of  fat,  depending  upon  whether  it  has  been  obtained 
from  whole  or  from  partially  or  more  completely  skimmed  milk. 

Ripening  of  Curd  into  Cheese. — The  ripening  of  the  curd  into  cheese 
consists  in  a  partial  or  more  or  less  complete  conversion  of  the  insoluble 
casein  into  simpler  and  soluble  proteid  or  albuminoid  bodies  and  in 
the  production  of  certain  bodies  which  give  to  the  ripe  cheese  the 
peculiar  taste  and  characteristic  flavor  which  vary  greatly  in  different 
varieties.  The  change  of  the  casein  which  resembles  that  brought 
about  by  gastric  juice  is  due  to  enzymes  secreted  by  bacteria  and 
other  microorganisms,  which  multiply  enormously  during  the  process 
of  ripening.  It  is  still  a  much  disputed  point  to  what  extent  certain 
bacteria  are  responsible  for  the  ripening  of  the  cheese.  It  is  believed, 
however,  by  many  that  the  hay  bacillus  (Bacillus  subtilis),  which 
secretes  a  strong  proteolytic  or  peptic  ferment,  plays  an  important  role 
in  the  conversion  of  casein  into  soluble  albuminoids.  In  some  soft 
cheeses  the  presence  of  Bacillus  subtilis  in  enormous  numbers  has 
been  established.  Ducleaux  has  shown  that  peptonizing  bacteria 
which  he  has  named  Tyrothrix  tenuis,  distortus  and  geniculatus,  are 
important  factors  in  the  ripening  of  certain  French  cheeses,  and 
Adametz  proved  the  presence  of  bacteria  likewise  endowed  with 
proteolytic  enzymes  in  soft  and  hard  Swiss  cheeses.  It  has  also  been 
shown  that  lactic-acid  bacteria  which  at  the  same  time  possess  the 
power  to  produce  certain  changes  in  casein  likewise  play  an  important 
role  in  the  ripening  of  cheese.  Liquefying  cocci  have  been  found  in 
cheese  by  von  Freudenreich.  Thoeni  and  Weigmann  and  Jensen 
have  shown  that  their  peptonizing  enzyme  is  an  important  factor  in 
the  early  changes  in  curd.  Von  Freudenreich  and  Jensen  have  also 
found  in  Swiss  cheese  anaerobic  bacteria  which  decompose  lactate  of 
lime  into  propionic  acid,  some  acetic  acid  and  carbon  dioxide,  and 
which  produce  holes  in  the  cheese,  improving  its  flavor  and  taste. 
Rodella  and  Weigmann  have  discovered  anaerobic  butyric  acid 
bacilli  in  Swiss  cheese  and  Paraplectrum  fcetidum  in  Limburger 
cheese.  Weigmann  gives  the  following  summary  of  the  activity  of 
bacteria  and  other  microorganisms  in  the  ripening  of  cheese: 

"The  lactic-acid  bacteria,  such  as  Streptococcus  Guentheri  and 
others,  which  remain  in  the  curd  with  the  larger  or  lesser  amount  of 
whey  contained  therein,  multiply  and  form  lactic  acid,  this  prevents 
the  immediate  activity  of  putrefying  bacteria  originally  contained 
in  the  milk  and  present  in  the  curd.  The  bacilli  most  susceptible 
to  acid,  such  as  the  Bacillus  coli,  some  of  the  hay  bacilli,  and  some 
of  the  anaerobics  present,  are  much  reduced  in  numbers,  liquefying 
cocci  and  lactic-acid  bacteria,  which  can  decompose  casein  without 
peptonization,  however,  multiply  considerably.  After  the  activity 


518  BACTERIA  IN.  BUTTER  AND  CHEESE-MAKING 

has  decreased  and  after  some  ammonia  has  been  formed,  hay  bacilli 
and  certain  hyphomycetes  become  more  active,  they  secrete  pepton- 
izing  enzymes,  and  the  ripening  process  makes  more  rapid  progress. 
The  lactose  fermentation  first  occurring  in  cheese  is  a  most  impor- 
tant process,  which  prevents  true  putrefactive  changes  and  prepares 
the  field  for  a  ripening  of  the  proper  kind.  Saccharomyces,  oidia 
and  hyphomycetes  probably  play  an  important  role  in  the  ripening  of 
cheese  and  in  giving  it  the  proper  flavor.  Saccharomyces  very  likely 
produce  esters  during  the  acid  formation;  oidia  and  hyphomycetes 
neutralize  the  acids  and  give  the  peptonizing  bacteria  a  chance  to 
multiply  and  to  produce  their  proteolytic  enzyme."  It  has  been  shown 
by  Conn  and  others  that  Camembert  cheese  owes  its  ripening  and  its 
flavor  and  taste  mainly  to  the  presence  of  Penicillium  candidum  and 
Penicillium  glaucum,  while  Roquefort  cheese  owes  its  properties  to 
the  presence  of  a  variety  of  Penicillium  glaucum(Penicillium  roqueforti 
or  Penicillium  aromaticum  casei).  The  bacteria  in  ripening  cheese, 
according  to  Conn,  for  a  number  of  days,  sometimes  for  several  weeks, 
increase  in  numbers.  After  this  they  decrease  until  when  the  cheese 
is  fully  ripened  they  are  very  few  compared  to  their  number  at  certain 
stages  of  the  ripening.  An  examination  showed  in  fresh  cheese 
6,600,000  bacteria  per  gram;  when  four  days  old,  51,000,000  per  gram, 
and  when  four  months  old,  1,000,000  per  gram.  Faults  in  cheese, 
such  as  a  gassy  condition,  swelling,  undesirable  flavor,  have  been  shown 
to  be  due  to  undesirable  bacteria  or  torulas.  To  prevent  such  faults 
Conn  recommends  cleanliness,  a  vigorous  lactic-acid  starter  for  the 
souring  of  the  milk,  and  a  temperature  of  60°  F.,  which  is  not  favorable 
to  those  organisms,  generally  responsible  for  the  faults  of  cheese. 


QUESTIONS 

1.  How  can  the  cream  be  separated  from  the  remainder  of  the  milk? 

2.  What  causes  the  ripening  of  the  cream? 

3.  How  much  acidity  does  ripe  cream  contain  ? 

4.  How  can  the  acidity  be  ascertained  ? 

5.  How  can  the  ripening  be  produced  by  the  addition  of  lactic  acid  ? 

6.  What  is  a  natural  starter? 

7.  What  is  an  artificial  starter? 

8.  What  microorganism  is  more  particularly  the  cause  of  the  ripening  of  the 
cream? 

9.  What  is  meant  by  aroma  microorganisms  ? 

10.  Why  is  their  use  sometimes  dangerous? 

11.  Discuss  the  advantages  of  using  pasteurized  cream  for  butter-making. 

12.  What  is  the  composition  of  buttermilk? 

13.  What  is  meant  by  the  building  up  of  the  starter? 

14.  WTiat  are  the  bacterial  contents  of  sweet  cream  and  of  ripe  (sour)  cream 

15.  Discuss  the  decrease  in  bacterial  content  during  butter-making  and  in  the 
butter  after  its  preparation. 

16.  What  is  rancid  butter;  what  causes  its  production  ? 

17.  What  are  the  proteids  contained  in  milk? 

18.  How  is  coagulation  of  milk  brought  about? 

19.  What  is  lab-enzyme  or  rennet? 

20.  How  can  it  be  obtained  ? 


, 


QUESTIONS 


519 


21.  What  is  known  about  bacterial  rennet? 

22.  Under  what  conditions  does  rennet  act? 

23.  What  is  meant  by  the  curd?    What  by  whey? 

24.  What  are  the  constituents  of  curd? 

25.  What  brings  about  the  change  from  curd  to  cheese  ? 

26.  What  are  the  essential  changes  in  the  ripening  of  cheese? 

27.  What  role  do  the  lactic-acid  bacteria  play  in  the  ripening  of  cheese  ? 

28.  What  is  the  role  of  liquefying  cocci? 

29.  What  of  peptonizing  bacteria? 

30.  What  of  moulds,  saccharomyces,  oidia? 

31.  What  is  meant  by  faults  of  cheese?    What  causes  them?, 

32.  How  can  they  be  prevented? 


CHAPTEE    XLIX. 

SIMPLE   CHEMICAL    MANIPULATIONS— NORMAL   SOLUTIONS   AND 

INDICATORS    REQUIRED    IN    LABORATORY    WORK 

IN    BACTERIOLOGY. 

REFERENCE  has  been  made  in  the  preceding  pages  to  certain 
simple  chemical  manipulations  and  tests  employed  in  standardizing 
the  reaction  of  culture  media,  estimating  the  change  in  their  reaction 
or  that  in  milk  in  consequence  of  bacterial  growth,  and  determining 
the  exact  amount  of  acid  or  alkali  formed.  These  manipulations 
require  a  set  of  simple  chemical  apparatus  and  a  few  chemical  reagents, 
standard  solutions,  indicators,  etc. 

Apparatus. — The  apparatus  required  is  the  following: 

1.  A  moderate-sized  balance  of  medium  delicacy,  carrying  about 
50  grams,  sensitive  to  1  milligram  or  less,  and  a  moderately  good  set 
of  weights  from  20  grams  to  1  milligram. 

2.  A  number  of  volumetric  flasks  holding  1000  c.c.,  500  c.c.,  250  c.c., 
100  c.c.,  and  50  c.c. 

3.  A  number  of  pipettes  holding  10,  5,  2  and  1  c.c.;  one  10  c.c. 
pipette  graduated  in  y1^  c.c.  and  one  1  c.c.  pipette  graduated  in  y^  c.c. 

4.  A  number  of  graduates,  beakers,  porcelain  evaporating  dishes, 
mortars,  flasks,  funnels,   test-tubes,  fermentation  tubes,  glass  and 
rubber  tubing,  and  glass  stirring  rods,  and  a  Kipp  gas  generator. 

5.  Several  burettes.   These  are  used  for  the  delivery  of  an  accurately 
measured  quantity  of  standard  or  normal  solutions.     A  burette  is 
made  from  a  long,  glass  tube  of  even  bore  throughout,  holding  50  or 
100  c.c.    On  the  glass  tube  lines  of  division  are  engraved,  correspond- 
ing generally  to  y^  c.c.    The  outlet  of  the  burette  is  either  a  rubber  tube 
with  clip  or  burette  clamp  and  a  glass  tip  or  a  ground-glass  stopcock. 
Burettes  are  used  in  an  upright  position,  held  in  a  burette  stand, 
and  the  fluids  from  them  are  generally  delivered  slowly,  drop  by  drop, 
by  manipulating  the  clip,  the  clamp,  or  the  stopcock. 

Gravimetric  and  Volumetric  Analysis. — The  method  of  determining 
quantitatively  a  chemical  substance  by  obtaining  it  first  in  a  pure 
state  or  in  a  compound  of  known  composition  and  then  weighing  it 
on  a  delicate  chemical  balance  is  called  the  gravimetric  method.  This 
method,  for  instance,  is  used  in  the  laboratory  in  determining  exactly 
the  amount  of  butter  fat  in  milk,  but  it  is  not  often  used  in  bacteriology. 
The  method  of  determining  quantitatively  the  amount  of  an  acid 
alkali  or  other  chemical  compound  by  the  aid  of  volumetric  or  standard 
solutions  is  called  volumetric  analysis,  and  this  is  the  method  generally 
employed  in  bacteriological  work. 


GRAVIMETRIC  AND  VOLUMETRIC  ANALYSIS  521 

Normal  Solutions. — A  normal  or  standard  solution  (N  sol.)  may  be 
defined  as  one  which  contains  one  molecular  weight  of  the  reagent 
in  grams  dissolved  in  enough  distilled  water  to  make  exactly  1000  c.c. 
at  a  temperature  of  16°  C.  (60°  F.).  If,  for  instance,  a  normal  solution 
of  caustic  soda  or  sodium  hydrate  (used  in  standardizing  our  culture 
media)  is  to  be  prepared  the  molecular  weight  of  sodium  hydrate, 
which  has  the  chemical  formula  NaHO,  must  first  be  ascertained.  The 
atomic  weight  of  sodium  (Na)  is  23,  that  of  hydrogen  (H)  is  1,  and 
that  of  oxygen  (O)  is  16.  Hence,  NaHO  has  a  molecular  weight  of  40. 
In  preparing  a  standard  or  normal  solution  of  NaHO,  therefore, 
40  grams  of  the  chemically  pure  sodium  hydrate  (NaHO  chemically 
pure)  are  weighed  out,  placed  in  a  1000  c.c.  volumetric  flask  and  dis- 
solved in  several  hundred  c.c.  of  distilled  water;  the  solution  (which 
has  become  warm)  is  allowed  to  cool  and  then  enough  distilled  water 
is  added  to  make  up  exactly  1000  c.c.  at  16°  C.  Sodium  hydrate  is 
a  hygroscopic  substance,  that  is,  one  which  will  draw  water  from  the 
atmospheric  air,  and  in  order  to  obtain  an  accurate  normal  solution 
it  must  be  weighed  out  rapidly.  In  fact,  in  exact  chemical  work  it  is 
impossible,  in  preparing  normal  solutions,  to  begin  with  a  normal 
solution  of  sodium  hydrate,  but  another  normal  solution  must  first  be 
prepared  from  a  substance  which  is  not  hygroscopic  and  which  can  be 
weighed  out  very  accurately.  Dry,  normal  sodium  carbonate,  Na2CO3, 
is  generally  used  for  this  purpose.  Another  factor  must  be  considered 
in  the  preparation  of  normal  solutions.  The  molecular  weight  in  grams 
(in  the  above  case  of  NaHO,  40  grams)  is  to  be  taken  only  in  case  of 
univalent  substances,  which  contain  one  hydrogen  atom  in  the  molecule, 
while  in  the  case  of  bivalent  substances,  which  contain  two  hydrogen 
atoms,  one-half  of  the  molecular  weight  in  grams  is  taken.  If  a 
normal  solution  of  sulphurie  acid,  H2SO4  (the  molecular  weight  of 
which  is  98),  is  to  be  prepared,  49  grams  are  taken  and  diluted  with 
enough  distilled  water  to  make  1000  c.c.  at  16°  C.  The  amounts  of 
the  reagents  to  be  taken  in  the  preparation  of  the  few  normal  solutions 
used  in  bacteriologic  work  are  as  follows : 

Grams  per 
1000  c.c. 

Crystallized  oxalic  acid 63.00 

Sulphuric  acid 49.00 

Hydrochloric  acid 36.37 

Nitric  acid 63.00 

Water-free  sodium  carbonate 53.00 

Sodium  hydrate 40.00 

Potassium  hydrate 56.00 

The  student,  however,  must  not  suppose  that  all  of  these  normal 
solutions  can  be  obtained  by  simply  weighing  out  the  amount  indicated 
and  dissolving  it  in  sufficient  water  to  make  1000  c.c.  Several  of  the 
above  chemicals,  sulphuric  acid  and  sodium  and  potassium  hydrate, 
for  instance,  are  very  hygroscopic ;  others,  like  nitric  acid  and  hydro- 
chloric acid,  evaporate  and  change  during  manipulation;  in  fact,  the 


522  SIMPLE  CHEMICAL  MANIPULATIONS 

only  substance  which  can  immediately  be  safely  used  to  obtain 
accurate  normal  solutions  is  the  perfectly  dry,  absolutely  pure  sodium 
carbonate,  Na2CO3.  Of  this,  53  grams  are  weighed  out,  placed  in 
a  1000  c.c.  volumetric  flask,  dissolved  with  several  hundred  cubic 
centimeters  of  distilled  water,  and  made  up  to  1000  c.c.  at  16°  C.  The 
next  solution  to  be  prepared  is  one  of  sulphuric  or  hydrochloric 
acid.  This  must  be  done  in  such  a  manner  that  1  c.c.  of  the  alkaline 
carbonate  of  sodium  solution  will  completely  neutralize  1  c.c.  of  the  acid 
solution,  and  vice  versa.  This,  however,  is  very  difficult  in  practice, 
and  generally  there  is  a  slight  discrepancy.  After  much  manipulation 
in  the  preparation  of  the  acid  normal  solution  it  may,  for  instance, 
be  found  that  49.8  c.c.  of  the  latter  will  just  neutralize  50  c.c.  of  the 
alkaline  normal  solution.  Such  a  slight  difference  can  be  entirely 
neglected  in  bacteriological  work;  in  very  exact  chemical  work, 
however,  correction  of  the  result  would  be  necessary  by  multiplication 
of  the  number -of  cubic  centimeters  used  out  of  the  burette  contain- 
ing the  slightly  too  strong  acid  normal  solution  by  T|\  =  100.4. 
As  a  rule,  normal  solutions  are  not  used  in  full  strength,  but  they 
are  diluted  with  distilled  water  in  proportions  of  1  in  2,  1  in  4, 1  in  5, 
1  in  10,  1  in  20, 1  in  100.  Such  dilutions  are  called  one-quarter  normal 
solution,  decinormal  solution,  centinormal  solution,  etc.,  and  desig- 
nated in  writing  as  ^  T>  TIP  ~2T>  TTO  >  e^c-  ^n  bacteriology  decinormal 
solutions  (YQ  sol.)  are  generally  used  and  prepared  by  taking  100  c.c. 
of  the  full  strength  normal  solution,  pouring  it  into  a  1000  c.c.  volum- 
etric flask  and  making  it  up  with  distilled  water  to  1000  c.c.  at  16°  C. 

When  a  normal  or  dilute  normal  solution  is  used  to  find  out  the 
exact  amount  of  a  certain  substance  in  another  solution  the  latter 
solution  is  said  to  be  titrated.1  In  doing  so  the  normal  solution  is 
allowed  to  flow  gradually  from  a  burette  into  an  exactly  measured 
amount,  say  10,  20,  or  50  c.c.  of  the  solution  which  is  being  tested. 
The  steps  of  such  a  titration  have  been  explained  in  detail  on  page  502 
in  the  chapter  on  Milk  in  the  Quantitative  Estimation  of  Lactic  Acid. 
A  titration  may  also  be  made  by  placing  a  definite  amount  of  the 
normal  solution  in  a  beaker  and  filling  the  other  solution  which  con- 
tains the  substance  to  be  determined  quantitatively  into  a  burette  and 
allowing  it  to  discharge  gradually  into  the  normal  solution  until  the 
reaction  is  complete. 

Indicators. — Whenever  a  titration  is  made  there  must  be  something 
to  indicate  when  the  reaction  is  complete.  If,  for  instance,  a  fluid  is 
titrated  for  the  amount  of  acid  which  it  contains  it  is  necessary  to 
know  when  enough  of  the  normal  alkaline  solution  has  been  added 
to  neutralize  the  acid  present.  A  reagent  added  to  the  acid  solution 

1  The  words  titrate  and  titration  are  derived  from  the  French  word  "titre,"  which  means 
title,  power,  or  strength.  Titrating  means,  therefore,  to  find  out  the  strength  or  concentration 
of  a  substance  in  a  solution.  This  word  is  also  used  a  good  deal  in  serum  investigations,  as, 
for  instance,  to  ascertain  the  titre  of  an  immune  serum,  etc. 


INDICATORS  523 

which  will  not  interfere  with  it  in  any  shape  or,  form  but  which  will 
tell  when  the  acid  has  been  completely  neutralized,  or,  rather,  when 
a  very  small  amount  of  the  alkaline  solution  has  been  added  in 
excess,  is  called  an  indicator.  In  general,  therefore,  an  indicator  is  a 
substance  which  by  a  change  of  color  or  a  precipitate  formed  or  in 
some  other  visible  manner  will  indicate  the  end  point  of  a  reaction. 
It  is  always  best  to  use  indicators  with  daylight  illumination,  because 
artificial  light  frequently  makes  the  color  reaction  less  characteristic, 
and,  therefore,  confusing. 

The  following  are  the  formulae  for  some  of  the  most  commonly 
employed  indicators  used  in  the  titration  of  acids  and  alkalies : 

Dimethylamidoazobenzol. — This  is  a  coal-tar  derivative  (anilin  stain) 
and  is  used  in  the  proportion  of  0.05  gr.  in  100  c.c.  of  95  per  cent, 
alcohol.  It  is  yellow  in  neutral  and  alkaline  solutions  and  red  in  acid 
solutions.  It  is  particularly  useful  in  the  titration  of  strong  mineral 
acids,  and  is  generally  used  in  the  determination  of  hydrochloric  acid 
(in  gastric  juice,  natural  or  artificial,  in  investigating  the  effect  of 
gastric  juice  upon  pathogenic  bacteria).  A  few  drops  of  the  indicator 
are  added  to  10  c.c.  of  the  gastric  juice.  The  fluid  in  the  presence 
of  HC1  assumes  a  red  color,  y^  sol,  NaHO  is  then  added  from  a 
burette  and  each  c.c.  of  the  normal  solution  used  in  neutralization 
is  equal  to  0.00365  gram  of  HC1. 

Cochineal. — This  substance  is  prepared  from  the  cochineal  louse 
(Coccus  cacti  cochinelifera),  living  on  certain  species  of  cactus.  It  is 
the  substance  from  which  the  carmine  used  for  staining  tissues  is 
also  derived.  Three  grams  of  cochineal  are  extracted  in  the  cold  with 
250  c.c.  of  25  per  cent,  alcohol.  This  cochineal  tincture  assumes  a 
violet  color  in  the  presence  of  alkalies  and  a  yellow-red  color  in  acid 
solutions.  It  is,  like  diamethylamidoazobenzol,  used  in  the  titration 
of  strong  mineral  acids. 

Litmus. — This  substance  is  of  vegetable  origin  and  derived  from 
several  species  of  lichens.  It  is  sold  in  commerce  in  the  form  of  small 
cubes  or  larger  cakes.  In  order  to  prepare  a  good  indicator  used  for 
general  laboratory  purposes,  or  for  the  preparation  of  litmus-lactose 
or  litmus-glucose  agar  and  gelatin,  it  is  necessary  to  boil  the  cubes 
with  three  or  four  changes  of  95  per  cent,  alcohol.  This  extracts  a 
dirty  violet  substance  from  the  commercial  article.  After  purification 
with  alcohol  the  cubes  are  soaked  in  water  until  the  latter  assumes 
a  dark  blue  color.  The  fluid  is  then  drawn  off  and  dilute  sulphuric 
acid  is  added  to  it  until  a  deep  violet  color  is  produced.  In  order  to 
secure  the  proper  color  it  is  necessary  to  take  a  few  c.c.,  dilute 
strongly  with  distilled  water,  and  examine  in  a  test-tube.  When  the 
dilute  fluid  has  a  reddish-violet  tint,  almost  a  cherry  red,  a  sufficient 
quantity  of  sulphuric  acid  has  been  added  to  the  original  watery 
extract.  Litmus  tincture  or  paper  is  stained  blue  in  alkaline,  red  in  acid 
solutions.  A  tincture  stained  slightly  blue  in  an  alkali  on  standing 
frequently  turns  red,  and  it  therefore  must  be  made  blue  again  before 


524  SIMPLE  CHEMICAL  MANIPULATIONS 

use  by  the  addition  of  alkali.  Litmus  is  the  most  commonly  used 
indicator  for  various  acids  and  alkalies. 

Rosolic  Acid,  or  Corallin. — This  is  an  aniline  stain.  It  is  dissolved 
in  the  proportion  of  0.5  gr.  to  50  c.c.  of  95  per  cent,  alcohol.  After 
solution  50  c.c.  of  distilled  water  is  added.  The  indicator  is  yellow  in 
neutral  and  acid  solutions  and  turns  rose  red  in  alkaline  solutions. 
It  is  particularly  useful  in  the  titration  of  organic  acids,  and  is,  there- 
fore, used  in  the  determination  of  lactic,  butyric,  formic,  succinic,  and 
acetic  acids. 

Phenolphthalein. — This  is  a  coal-tar  derivative,  and  is  very  much 
used  in  work  in  bacteriology.  It  is  generally  employed  in  the  exact 
titration  of  culture  media,  as  previously  explained  in  detail.  The 
indicator  is  prepared  as  a  1  per  cent,  alcoholic  solution.  It  is  colorless 
in  neutral  and  acid  solutions  and  turns  red  in  alkaline  solutions. 
The  change  of  color  in  this  solution  can  also  be  well  seen  by  artificial 
illumination.  It  also  takes  place  promptly  in  hot  fluids,  and  the 
phenolphthalein  indicator  has,  therefore,  a  wide  range  of  application. 
It  can,  however,  not  be  depended  upon  for  the  determination  of 
ammonia  and  the  weak  alkalies,  but  it  is  excellent  for  the  hydrates  of 
sodium,  potassium,  calcium,  and  barium. 

Empirical  Standard  Solution. — These  are  based  upon  a  different 
principle.  They  are  not  prepared  according  to  the  molecular  formula 
of  the  chemical  compound  employed,  but  in  such  a  manner  that  1  c.e. 
of  the  standard  solution  will  be  equivalent  to  10  milligrams  of  the 
substance  which  is  to  be  determined  quantitatively  by  the  volumetric 
analysis. 

FEELING'S  STANDARD  SOLUTION. — A  fluid  of  this  type  is  Fehling's 
standard  solution  for  the  quantitative  determination  of  sugars  (glucose, 
dextrose,  maltose,  lactose).  As  the  quantitative  determination  of 
sugar,  either  to  ascertain  how  much  of  it  has  been  fermented  by 
certain  bacteria  or  has  been  formed  from  starch,  frequently  must  be 
made  in  bacteriological  work,  the  formula  for  Fehling's  copper  solution 
and  the  steps  in  the  analysis  will  be  given  here. 

Fehling's  Standard  Solution: 

SOLUTION  1. 

Sulphate  of  copper,  chemically  pure  crystals 34.638  grams 

Sulphuric  acid,  chemically  pure 1  c.c. 

Dissolved  in  enough  distilled  water  to  make 500  c.c. 

SOLUTION  2. 

Tartrate  of  sodium  and  potassium,  chemically  pure    (Rochelle  salt)       175  grams 

Sodium  hydrate,  chemically  pure ....          125  grams 

Distilled  water  enough  to  make     .      .      .      .     ,      .-  .    .\     .      .  .       500  c.c. 

Two  cubic  centimeters  of  the  copper  and  alkaline  solution  mixed 
is  equivalent  to  10  milligrams  of  glucose  or  dextrose,  to  16  milligrams 
of  maltose,  and  to  13.5  milligrams  of  lactose.  Solutions  No.  1  and 
No.  2  must  be  kept  separate.  They  are  mixed  only  in  exactly  equal 


EMPIRICAL  STANDARD  SOLUTION  525 

proportions  just  before  use,  because  the  mixture  is  not  very  stable  and 
decomposes.  After  the  two  fluids  are  mixed  the  copper  is  present 
in  the  form  of  a  hydrate  of  the  metal,  and  this  hydrate  in  hot  alkaline 
solution  is  reduced  by  the  sugars  named  into,  first,  a  cupric  oxide,  and 
then  a  cuprous  oxide.  The  former  is  a  yellow,  the  latter  a  red-brown 
copper  salt.  The  details  of  this  process  of  reduction  are  not  yet 
perfectly  clear,  but  it  is  established  beyond  doubt  that  10  milligrams 
of  glucose,  16  of  maltose,  or  13.5  of  lactose  will  bring  about  the 
complete  reduction  of  all  of  the  copper  hydrate  contained  in  2  c.c. 
of  the  mixture  of  solutions  No.  1  and  No.  2;  that  is,  of  the  copper 
salt  originally  contained  in  1  c.c.  of  solution  No.  1.  When  the 
reduction  of  the  copper  hydrate  occurs  in  the  hot  alkaline  solution  a 
yellowish  or  orange  precipitate  is  generally  first  formed,  and  finally  a 
red-brown  precipitate  at  the  bottom  of  the  beaker;  the  supernatant 
fluid  after  the  fluid  has  become  cool  is  perfectly  colorless  (all  blue 
color  has  disappeared).  As  it  would  be  impossible  to  recognize  at  the 
right  moment  the  end  point  of  the  reaction,  i.  e.,  the  complete  reduction 
of  the  copper  hydrate)  an  indicator  must  be  used.  As  such  a  saturated 
watery  solution  of  ferrocyanide  of  potash  strongly  acidulated  with 
glacial  acetic  acid  is  employed  in  the  following  manner:  Small 
pieces  of  filter  paper  are  moistened  with  the  indicator  and  from  time 
to  time  a  drop  of  the  boiling  copper  solution  (to  which  the  sugar- 
containing  fluid  from  the  burette  is  being  added)  is  allowed  to  fall  on 
them.  As  long  as  unreduced  copper  hydrate  remains  in  the  boiling 
solution  it  forms  a  red  cyanide  of  copper  with  the  acid  ferrocyanide 
solution.  When  the  reduction,  however,  is  completed  no  more 
cyanide  of  copper  is  formed  and  no  more  sugar  containing  fluid 
should  be  added  from  the  burette. 

Steps  in  the  Determination. — 1.  Mix  equal  amounts  of  solutions 
from  bottle  No.  1  and  No.  2.  This  mixture  is  the  standard  solution 
now  ready  for  use. 

2.  Take  20  c.c.  of  the  mixture,  place  into  a  beaker,  dilute  with 
30  c.c.  of  distilled  water,  and  heat  over  a  small  flame,  or,  still  better, 
in  a  water  bath. 

3.  The  sugar  containing  fluid  must  first  be  filtered,  and  if  it  contain 
much  sugar  (which  should  be  ascertained  by  a  preliminary  qualitative 
test)  it  should  be  diluted  with  distilled  water,  so  that  it  probably 
contains  between  J  and  1  per  cent,  sugar  approximately. 

4.  Heat  the  standard  solution  in  the  beaker  to  boiling  and  then  add 
the  sugar  solution  gradually  from  the  burette.    Continue  heating  and 
adding,  and  from  time  to  time  test  the  boiling  solution  with  the  indi- 
cator.   When  cyanide  of  copper  ceases  to  be  formed  on  the  moistened 
filter  paper,  stop  adding  sugar  solution  from  the  burette.    If  the  test 
has  been  made  properly  the  fluid  in  the  beaker  should  be  colorless 
after  cooling,  and  if  mixed  with  the  indicator  should  not  form  a  red 
precipitate.    Nor  should  it  look  yellow,  for  this  would  indicate  that 
an  excess  of  sugar  solution  had  been  added. 


526  SIMPLE  CHEMICAL  MANIPULATIONS 

5.  After  the  completion  of  the  reaction,  read  off  from  the  burette 
the  number  of  c.c.  of  sugar  solution  used  and  calculate  from  this 
figure  the  percentage  of  sugar  present  in  the  original  sugar-containing 
fluid. 

Example. — As  an  example,  suppose  that  a  3  per  cent,  starch  bouillon 
has  been  inoculated  with  a  microorganism,  secreting  a  diastatic 
ferment,  and  that  the  amount  of  sugar  formed  after  three  days' 
incubation  is  to  be  ascertained.  The  solution  must  first  be  filtered. 
The  preliminary  qualitative  test,  also  made  with  Fehling's  solution, 
shows  considerable  sugar  present.  The  filtrate  is,  therefore,  diluted 
in  the  proportion  of  1  to  3  parts  of  distilled  water  and  the  fluid  so 
obtained  filled  into  a  clean  burette  in  which  it  is  so  regulated  that 
40  c.c.  are  present  when  the  addition  to  the  boiling  copper  solution 
is  begun.  When  all  the  copper  hydrate  has  been  reduced  it  is  found 
that  24  c.c.  have  been  used  out  of  the  burette.  This  amount  of  fluid 
contains  100  milligrams  of  sugar,  which  are  necessary  to  reduce  all  of 
the  copper  hydrate  in  20  c.c.  of  Fehling's  solution;  hence,  100  c.c.  .of  a 
fluid  like  the  one  used  in  the  burette  contain  416  milligrams.  Since 
the  fluid  in  the  burette  represents  one-fourth  of  the  concentration  of 
the  original  filtered  liquid,  the  latter  contains  four  times  as  much  sugar 
as  the  dilute  fluid  used  in  the  burette,  or  1664  milligrams=  1.664  grams 
of  sugar  per  100  c.c.,  or  1.664  per  cent.  If  n  is  the  number  of  c.c. 
used  out  of  the  burette  then  the  following  equation  results : 

n  :   100  =  100  :  x 
and  x  =  10,000 
n 

In  other  words:  To  find  the  amount  of  sugar  in  milligrams  present 
in  100  c.c.  of  the  fluid  used  in  the  burette,  divide  10,000  by  the  number 
of  c.c.  used  out  of  the  burette.  This  gives  the  amount  of  sugar  in 
milligrams  per  100  c.c.  for  the  dilute  fluid,  and  it  must  be  multiplied 
by  the  number  of  times  diluted  to  express  the  amount  of  sugar  in 
milligrams  present  in  100  c.c.  of  the  original  fluid.  The  sugar  in  this 
example  has  been  calculated  as  glucose  or  dextrose.  In  the  case  of 
maltose  the  division  is  made  into  16,000  in  the  same  manner  and  in 
the  case  of  lactose  into  13,500,  because  these  sugars  reduce  copper 
hydrate  in  a  different  manner.  Saccharose  does  not  reduce  copper 
hydrate,  and  when  it  is  to  be  determined  by  the  aid  of  Fehling's 
solution  it  must  first  be  changed  into  invert  sugar  by  boiling  with 
dilute  acids  or  by  the  action  of  the  enzyme  invertin. 


QUESTIONS  527 


QUESTIONS 

1.  What  apparatus  is  required  for  the  simple  chemical  manipulations  employed 
in  elementary  bacteriology? 

2.  What  is  a  burette,  a  pipette,  a  beaker,  an  evaporating  dish? 

3.  What  is  meant  by  the  gravimetric,  what  by  the  volumetric  method  of 
quantitative  chemical  analysis? 

4.  What  is  a  normal  solution  ?    What  a  decinormal,  centinormal  solution  ? 

5.  What  is  the  atomic  weight  of  an  elementary  body?    What  is  the  molecular 
weight  of  a  compound  body  ? 

6.  What  is  the  atomic  weight  of  N,  O,  C,  and  S? 

7.  What  is  the  molecular  weight  of  caustic  soda? 

8.  What  is  the  procedure  in  preparing  a  set  of  acid  and  alkaline  normal 
solutions  for  use  in  the  standardization  of  the  common  culture  media? 

9.  How  is  the  acid  standardized  against  the  alkaline  solution?     Is  it  easy 
to  prepare  them  so  that  100  c.c.  of  the  acid  normal  solution  will  exactly  neutralize 
100  c.c.  of  the  alkaline  normal  solution? 

10.  If  they  are  not  exactly  balanced  what  can  you  do  to  correct  the  result  ? 

11.  What  is  a  titration? 

12.  What  is  an  indicator? 

13.  Describe  the  preparation  of  the  following  indicators:     Dimethylamidoazo- 
benzol,  litmus,  rosolic  acid,  phenolphthalein. 

14.  Give  the  formula  for  Fehling's  standard  solution. 

15.  What  is  an  empirical  standard  solution? 

16.  Describe  the  use  of  Fehling's  standard  solution  in  the  quantitative  esti- 
mation of  sugar  in  a  fluid. 

17.  How  much  of  the  following  sugars  does  it  take  to  reduce  all  of  the  copper 
hydrate  contained  in  20  c.c.  of  Fehling's  solution?       Glucose,  maltose,  lactose? 

18.  Describe  the  indicator  used  in  connection  with  Fehling's  solution. 

19.  How  does  this  indicator  work  ? 


PART  IV. 
PATHOGENIC  PROTOZOA. 


CHAPTER    L. 

GENERAL  CONSIDERATION  OF  PROTOZOA— CLASSIFICATION- 
MORPHOLOGY  AND  REPRODUCTION. 

Definition  and  Morphology. — Protozoa  represent  the  lowest  and  most 
simple  form  of  animal  life.  The  organisms,  however,  vary  much 
in  their  morphology.  While  unicellular  and  occasionally  united  in 
colonies  of  unicellular  individuals,  they  may  have  a  body  with  a 
variety  of  parts,  or  small  organs  called  organella,  serving  different 
purposes.  In  this  respect  protozoa  when  compared  with  the  lowest 
forms  of  vegetable  life  (the  bacteria)  are  considerably  higher  in 
structural  differentiation.  In  a  summary  manner  protozoa  may  be 
defined  as  unicellular  animal  organisms.  Calkins  gives  the  following 
more  elaborate  definition :  "A  protozoon  is  a  primitive  animal  organ- 
ism, usually  consisting  of  a  single  cell,  whose  protoplasm  becomes 
distributed  among  many  free  living  cells.  These  reproduce  their 
kind  by  division,  by  budding,  or  by  spore  formation,  the  race  thus 
formed  passing  through  different  form  changes,  and  the  protoplasm 
through  various  stages  of  vitality  collectively  known  as  the  life  cycle." 

Protozoa,  like  the  lowest  forms  of  vegetable  life,  are  very  prevalent 
in  nature,  but  they  do  not  find  the  conditions  necessary  for  or  favor- 
able to  their  nutrition  and  multiplication  as  easily  as^the  bacteria.  The 
lowest  forms  of  animal  life  being  highly  differentiated  and  representing 
a  great  variety  of  morphologic  features,  the  phylum  protozoa  has 
been  divided  into  "several  subphyla,  numerous  classes,  subclasses, 
orders,  families,  and  genera. 

Protozoa,  like  bacteria,  live  either  in  the  outside  world  or  as  para- 
sites on  or  in  other  organisms.  They  may  exist  as  harmless  commen- 
sales  or  they  may  be  pathogenic  parasites. 

The  number  of  protozoa  known  to  be  pathogenic  to  man  and 

the  higher  animals  is  comparatively  small.     As  these  belong  to  a 

few  families  only,  medical  and  veterinary  studies  of  protozoa,  aside 

from  a  general  survey  of  the  phylum  protozoa,  may  be  confined  to 

34 


530  GENERAL  CONSIDERATION  OF  PROTOZOA 

the  morphology  and  the  biologic  characters  of  a  very  limited  number 
of  families. 

Shape  and  Size. — Protozoa,  composed  as  they  are  of  a  soft  proto- 
plasm, present  themselves  under  various  shapes,  depending  upon 
differences  in  environment;  they  may  be  more  or  less  spherical,  or 
homaxonic,  or  they  may  be  decidedly  elongated  in  one  axis,  or  mon- 
axonic.  When  the  environments  become  unfavorable  and  under 
other  conditions,  protozoa  often  become  perfectly  spherical,  and 
secrete  a  thick,  resistant  membrane  composed  of  chitin.  This  change 
is  known  as  the  encysted  stage.  The  organism  may  die  in  it  or  it  may 
relinquish  the  encysted  condition  and  return  to  its  former  shape, 
divide  under  the  protecting  membrane  into  daughter  cells,  or  sporulate 
when  circumstances  become  more  favorable.  Protozoa  are  evidently 
more  resistant  in  the  encysted  stage,  and  it  may  in  certain  respects 
be  likened  to  the  spore  formation  in  bacteria,  though  spore  formation 
in  protozoa  is  a  process  quite  different  and  distinct  from  encysting. 
As  a  class  of  organisms  protozoa  vary  so  much  in  shape  that  no 
common  description  will  fit  all  of  them.  They  likewise  vary  greatly 
in  size  (from  one  or  a  few  micra  to  several  millimeters),  and  it  cannot 
even  be  said  that  all  protozoa  are  microorganisms,  as  some  can  be 
easily  seen  with  the  naked  eye.  Porospora  gigantea,  a  gregarina 
found  in  the  intestines  of  the  lobster,  is  16  mm.  long.  The  individual 
organisms  of  one  and  the  same  species  will  also  frequently  vary 
considerably  in  size,  much  more  than  bacteria.  This  variability 
often  depends  upon  the  amount  of  available  food  supply  and  upon 
other  external  conditions. 

Structure. — Bacteria  do  not  yet  show  a  differentiation  of  the  cell 
into  a  protoplasmic  body  and  a  nucleus ;  all  protozoa,  however,  possess 
a  distinct  nucleus  or  several  nuclei.  The  cell  protoplasm  or  cytoplasm 
of  protozoa  is  composed  of  the  spongioplasm,  which  generally  has  a 
network  or  honey-combed  structure,  and  is  made  up  of  a  rather  firm, 
tenacious  substance,  and  the  hyaloplasm,  which  is  a  more  fluid 
substance  contained  in  and  filling  out  the  network  of  the  spongio- 
plasm. The  outer  layer  of  the  protozoan  organism  generally  is 
composed  of  a  more  condensed  and  tougher  protoplasm  called  the 
ectoplasm  in  contradistinction  to  the  softer  protoplasm  within  known 
as  the  entoplasm.  The  ectoplasm  sometimes  secretes  or  forms  a 
membrane,  an  armor,  organs  of  locomotion,  etc.  Round  or  oval 
spaces  called  vacuoles  are  frequently  seen  inside  of  the  protoplasm. 
They  are  not  empty  or  air-containing  spaces,  but  are  filled  with  a 
watery  fluid  and  are  either  concerned  in  the  digestion  and  assimilation 
of  food  particles  or  are  contractile  vacuoles  which  empty  and  refill. 
Protozoa  sometimes  contain  a  complicated  system  of  intercommuni- 
cating vacuoles  through  which  fluid  more  or  less  constantly  circulates. 
In  addition  to  the  vacuoles  the  cytoplasm  of  protozoa  shows  granules 
of  various  size  and  shape.  Thus  the  small  granules  of  Altmann, 
supposed  to  be  intimately  connected  with  the  ultimate  structure  and 


ORGANS  OF  LOCOMOTION  531 

function  of  the  protoplasm  as  such  are  found;  also  food  particles, 
pigment  granules,  oil  drops,  waste  material  and  foreign  material,  like 
lime  or  silica,  more  or  less  accidentally  taken  up  into  the  body  of  the 
organism.  The  granules,  which  contain  stored  food  material,  are 
often  called  plastids,  and  plastids  containing  pigment  are  known  as 
chromatophores.1 

Nucleus  and  Nuclear  Substances. — Protozoa  generally  contain  two 
nuclei.  In  some  protozoa  these  cannot  be  well  recognized  as  two 
distinct  nuclei,  because  they  are,  during  the  period  of  rest,  contained 
in  one  common  nuclear  membrane;  in  other  protozoan  organisms 
they  can  always  be  well  distinguished.  Since  one  of  the  nuclei  is 
generally  large  and  the  other  small,  they  have  been  called  the  macro- 
nucleus  and  the  micronucleus;  the  latter  is  also  called  the  blepliaroblast, 
because  a  flagellum  may  originate  from  it.  The  large  nucleus  is 
generally  concerned  in  the  nutrition  of  the  organism;  hence  it  is 
called  the  trophonucleus.  The  small  or  micronucleus  is  clearly 
concerned  in  the  reproduction  and  multiplication  of  the  protozoan 
organism,  and,  for  this  reason,  Calkins  recommends  that  it  be  called 
the  karyogonad,  or  the  gonad  nucleus,  as  representing  the  germ  plasm 
from  which  reproduction  starts.  This  gonad  nucleus,  when  contained 
in  a  common  nuclear  membrane  with  the  trophonucleus  separates 
from  it  during  the  period  of  maturation  which  precedes  reproduction. 
That  portion  of  the  nucleus  which  stains  with  the  so-called  nuclear 
stains  (hematoxylin,  carmin,  the  basic  anilin  stains)  is  called  the 
chromatin.  The  latter  under  certain  conditions  may  leave  the  nucleus 
and  become  distributed  diffusely  in  the  form  of  granules  in  the 
cytoplasm.  Besides  the  chromatin  the  nuclei  of  protozoa  contain 
another  very  important  substance,  which  apparently  is  the  source  of 
energy  of  motion  and  metabolism;  this  substance  has  been  called 
kinoplasm  (also  archoplasm).  Calkins  calls  this  kinoplasm,  whether 
found  inside  or  outside  the  nucleus,  the  division  centre.  Its  importance 
in  relation  to  the  function  of  locomotion  is  well  recognized  in  trypano- 
somes  where  the  material  of  the  undulating  membrane,  the  flagellum 
and  the  other  contractile  locomotory  structures,  is  all  derived  from 
this  division  centre,  or  kine  to  nucleus. 

Organs  of  Locomotion. — 1.  Pseudopodia. — The  most  primitive  form 
of  locomotion  among  protozoa  is  the  ameboid  motion,  depending 
upon  the  possession  of  pseudopodia  (false  feet).  These  are  simply 
digit-like  prolongations  or  out-flowings  of  the  cytoplasm.  In  ameba 
the  latter  under  favorable  conditions  is  constantly  in  a  flowing  motion 
combined  with  a  constant  change  of  shape.  This  ameboid  motion 
by  the  flowing  and  drawing  action  of  the  contractile  protoplasm  is 
not  only  shown  by  certain  protozoa,  but  also  by  some  of  the  cells  of 
higher  multicellular  (metazoic)  animals.  The  most  important  cells 

1  The  term  chromatophore  in  histology  and  histopathology  of  higher  animals  designates 
pigment  containing  cells  and  not  mere  plastids. 


532       GENERAL  CONSIDERATION  OF  PROTOZOA 

of  this  type  are  certain  white  blood  corpuscles,  or  leukocytes.  Just 
as  these  can  send  out  pseudopodia,  crawl  around  in  the  tissues  and 
engulf  and  digest  bacteria,  so  certain  protozoa  known  as  amebce  exhibit 
exactly  the  same  phenomena.  A  distinction  is  made  between  lobopodia 
which  are  the  digit-like,  irregular,  soft,  and  inconstant  pseudopodia, 
and  filopodia,  which  are  more  stiff,  less  motile,  and  quite  permanent. 
They  are  often  distributed  more  or  less  regularly  around  the  body  of 
certain  protozoon  organisms,  and  are  therefore  known  as  actinopodia 
(star  feet). 

2.  Flagella. — In  protozoa   these  are   tapering  filaments,  broadest 
at  their  attached  base  and  ending  in  a  fine  tip.    Protozoa  may  have 
a  flagellum  at  the  anterior  or  at  the  posterior  end;  they  may  have  a 
flagellum  at  each  end  and  a  large  number  of  them  distributed  entirely 
around  the  body.    Dobell  distinguished  four  types,  depending  upon 
the  origin  of  the  flagellum :    One  in  which  the  flagellum  arises  directly 
from  the  nucleus;  a  second,  in  which  the  flagellar  base  is  united  to 
the  nucleus  by  a  connecting  filament,  the  zygoblast;  a  third,  in  which 
the  flagellum  arises  from  a  basal  granule,  which  is  independent  of 
the  nucleus,  as  in  herpetomonas ;  and  a  fourth,  in  which  the  flagellum 
arises  from  a  special  kinetonucleus  (blepharoblasf),  as  in  trypanosoma. 

3.  Cilia. — The  cilia  of  protozoa  are  similar  in  type  to  the  cilia  lining 
the  epithelial  cells  of  the  nasal  or  uterine  mucosa  in  higher  animals. 
They  are  generally  shorter  than  flagella,  and  like  them  broader  at  the 
base  and  pointed  at  the  distal  end.    They  are  peculiar  in  their  motion; 
first  they  move  rapidly  and  energetically  in  one  direction,  and  then 
they  withdraw  slowly  to  the  opposite  direction.    This  double  motion 
is  repeated  more  or  less  continually.    Cilia  generally  show  a  granule 
at  their  basal  attached  end.     They  may  be  distributed  uniformly 
over  the  whole  external  surface,  limited  to  one-half  or  to  the  ventral 
surface  of  the  body  of  the  protozoon,  or  arranged  in  a  single  circle 
around  the  mouth  opening.     In  some  cases  cilia  are  united  together 
to  a  brush,  in  other  cases  they  are  completely  fused  to  membrane  or 
leaf-like  masses,  which  are  then  called  membranelles.     The  basal 
granules  from  which  cilia  arise  have  a  nuclear  derivation,  and  are 
called  microsomes .     In   many   infusoria  such  kinetic  granules   are 
arranged  in  threads  and  rows.     They  form  a  contractile  substance, 
evidently  related  to  the  muscle  substance  of  higher  animals,  and  these 
primitive  muscles  are  called  myonemes. 

Reproduction. — It  has  been  seen  that  bacteria  which  have  no  differ- 
entiated nuclei  multiply  simply  by  binary  division  or  fission.  The 
blastomycetes,  yeast  cells,  or  budding  fungi,  which  are  somewhat 
higher  in  phylogenetic  development  among  the  simplest  forms  of 
vegetable  life,  possess  a  nucleus,  and  when  they  multiply  by  spore 
formation  or  budding  a  division  of  the  nucleus  always  occurs,  with 
a  division  of  the  cytoplasm.  All  animal  cells  possess  nuclei,  and 
nuclear  division  always  occurs  in  cell  reproduction  or  multiplication. 
When  the  nucleus  simply  divides  as  a  whole  without  the  formation 


MODES  OF  SEXUAL  REPRODUCTION  IN  PROTOZOA        533 

of  a  special  characteristic  arrangement  of  the  chromatic  substance 
the  process  is  called  amitotic  division;  when  the  chromatin  arranges 
itself  in  a  very  definite  manner  in  bands  or  rods,  which  become  equally 
divided,  the  process  is  known  as  mitotic  division,  karyokinesis ,  or 
karyomitosis.  When  this  form  of  division  (quite  common  in  protozoa) 
occurs  the  kinetic  achromatic  substance  of  the  nucleus  arranges 
itself  in  the  form  of  an  achromatic  spindle,  with  a  very  small  granule 
or  point,  the  centrosome,  at  either  end.  The  chromatic  substance 
at  the  same  time  forms  a  definite  number  of  threads,  bands  or  rods, 
which  divide  by  splitting  into  double  the  number  originally  present. 
They  then  move  in  equal  numbers  toward  the  centrosomes,  so  that 
at  the  end  of  division  each  nucleus  possesses  again  the  same  number 
of  chromosomes  as  the  original  nucleus  when  it  was  ready  to  divide 
and  its  membrane  began  to  be  dissolved.  The  number,  type,  and 
character  of  chromosomes  is  the  most  constant  morphologic  part  of 
a  cell,  and  all  cells  of  the  same  species  have  the  same  number  of 
chromosomes  which  are  considered  as  the  carriers  of  all  the  elements 
transmissible  by  heredity.1  When  two  germ  cells,  however,  unite  to 
form  a  new  being  the  number  of  chromosomes  in  each,  by  a  process 
of  maturation  and  by  the  expulsion  of  chromosomes  into  polar  bodies, 
becomes  reduced  to  one-half,  so  that  the  new  cell  formed  by  the 
union  of  two  mature  germ  cells  has  not  double  the  original  constant 
number  of  chromosomes.  Several  types  of  cell  division  occur  among 
protozoa.  There  is  simple  binary  division  with  splitting  into  two 
equal  parts  or  new  cells.  This  is  the  type  generally  found  among 
the  cells  of  higher  metazoic  animals.  Budding  as  seen  in  the  budding 
fungi  or  yeast  cells  is  also  encountered,  or  there  may  be  a  splitting  up 
of  the  cell  into  a  number  of  smaller  daughter  cells,  each  receiving  its 
proportionate  share  of  the  nucleus.  The  nuclear  changes  in  protozoa 
may  closely  resemble  and  be  as  complicated  as  the  karyokinesis  in 
higher  cells,  or  the  granular  chromatin  may  divide  as  such  without 
solution  of  the  nuclear  membrane,  which  may  simply  become  con- 
stricted in  the  middle  and  finally  be  cut  in  two  at  this  point.  The 
cells  of  higher  animals  when  dividing  generally  assume  a  simple 
globular  form,  but  in  protozoa  division  is  found  in  fully  differen- 
tiated cells,  for  instance,  in  the  flagellate  trypanosomes.  Starting 
with  the  blepharoblast  (micronucleus,  root  of  the  flagellum)  all  the 
structures  of  the  organism,  including  the  flagellum,  undulating 
membrane,  macronucleus,  and  cytoplasm,  become  split  in  two  some- 
times equal,  sometimes  unequal,  masses. 

Different  Modes  of  Sexual  Reproduction  in  Protozoa. — In  the  phylum 
protozoa  a  variety  of  types  of  reproduction  are  encountered. 

1.  Autogamy,  or  Automyxis. — In  the  fertilization  by  autogamy,  or 
automyxis,  which  is  widespread  among  protozoa,  there  occurs  an 

1  It  is,  however,  almost  certain  that  the  cytoplasm  likewise  contains  certain  constant  elements 
always  propagated  in  multiplication,  which  are  likewise  the  carriers  of  hereditary  properties 
of  the  living  substance. 


534 


GENERAL  CONSIDERATION  OF  PROTOZOA 


expulsion  of  chromatin  from  the  nucleus  into  the  cytoplasm.  The 
cromidia,  or  idiochromidia,  so  formed  collect  in  more  or  less  well-defined 
masses,  and  these  are  known  as  the  secondary  nuclei,  which,  however, 
have  no  nuclear  membranes,  but  consist  simply  of  seggregated  masses 
of  extranuclear  chromatin.  Two  such  masses  fuse,  and  this  is  the 
process  of  syngamic  nuclear  union,  autogamy,  or  automyxis.  It  is  a 
self-fertilization  and  the  most  primitive  type  of  sexual  reproduction 

FIG.  182 


Amoeba  limax.     Chromidia  forming  from  nucleus  and  collecting  in  the  cytoplasm  prior  to 

encystment.     (Calkins.) 

in  protozoa.  It  occurs  in  many  species  of  ameba,  and  has  been 
studied  frequently  in  Amoeba  limax.  In  Entamceba  hystolytica,  the 
parasite  causing  amebic  dysentery  in  man  and  monkeys,  which  is 
not  only  prevalent  in  the  tropics  but  also  in  the  southern  parts  of 
this  country,  and  even  sporadically  in  the  Northern  States,  Schaudinn 
and  Craig  have  observed  the  formation  of  idiochromidial  masses  by  a 
fragmentation  of  the  original  nucleus.  These  masses  become  located 
at  the  periphery,  are  provided  with  some  cytoplasm  from  the  surface 

FIG.  183 


Amoeba  limax  budding,  division,  and  idiochromidia  forming  stages.     (Calkins.) 

as  buds,  and  are  finally  cut  off.  In  this  species  of  ameba,  however, 
the  union  of  two  masses  of  idiochromidia  has  not  been  actually 
observed.  Other  species  of  ameba  show  a  more  complicated  process 
of  chromidia  formation  and  union  (conjugation)  between  two  masses 
formed.  In  Amoeba  proteus,  Calkins  has  observed  the  following 
process  of  autogamous  reproduction.  There  is  no  formation  of 
diffused  idiochromidia,  but  the  secondary  conjugating  nuclei  are 


MODES  OF  SEXUAL  REPRODUCTION  IN  PROTOZOA       535 

formed  directly  from  chromatin  granules  within  the  primary  nucleus, 
which,  prior  to  this  stage,  had  divided  repeatedly  until  about  70  are 
present.  These  secondary  nuclei  next  fuse  2  by  2  in  the  cytoplasm 
and  give  rise  to  spore  mother  cells  (sporoblasts),  of  which  there  may 
be  as  many  as  250  writhin  one  parent  organism,  while  at  least  one  of 
the  primary  nuclei  remains  unused  and  finally  degenerates  in  the  cell. 
In  Amoeba  proteus,  therefore,  in  autogamous  fertilization  the  organ- 
ism does  not  form  one  spore-mother  cell  (sporoblast),  but  many. 

2.  Endogamy. — In  the  mode  of  fertilization  known  as  endogamy  the 
cell  protoplasm  breaks  down  into  a  number  of  portions,  each  one  of 
which  receives  some  nuclear  material.    After  the  division  of  the  proto- 
plasm has  taken  place  two  of  the  distinct  and  separated  masses  fuse, 
and  around  the  two  united  gametes,  now  known  as  a  copula,  a  spore 
wall  is  formed. 

3.  Exogamy. — When  two  cells  from  different  ancestors  unite  and 
become  completely  fused  the  process  is  known  as  exogamy.     This 
method  is  very  much  like  reproduction  in  higher  metazoic  animals, 
where  the  male  and  female  germ  cells  become  fused  to  form  a  new 
cell.    The  copulating  protozoan  cells  may  be  perfectly  alike  (isogamy), 
or  they  may  be  different  in  type  (anisogamy).    In  the  latter  case,  in 
which  two  cells  different  in  morphology  become  united,  one  can  be 
likened  to  the  male  and  the  other  to  the  female  germ  cells  of  metazoa. 
These  germ  cells  of  protozoa  are  called  gametes.     Budding  is  inti- 
mately  associated  with  conjugation,   the   buds   are   supplied   with 
chromatin,    and   they  often   subsequently  become   the   conjugating 
gametes.     Budding,  however,  differs  from  spore  formation.    In  the 
former  case  the  mother  organism  which  gives  off  the  buds  continues 
to  live,  while  in  spore  formation  the  mother  cell  breaks  up  into  spores 
and  ceases  to  exist. 

Spore  Formation. — In  some  protozoa,  particularly  in  many  flagellata, 
this  follows  conjugation  of  two  similar  cells  (isogamy).  These 
similar  cells,  after  having  united,  form  a  common  cyst,  and  the  proto- 
plasm in  the  interior  of  the  latter  becomes  split  into  a  great  many 
very  small  flagellated  organisms.  Among  the  class  of  protozoa  called 
sporozoa  there  are  two  types  of  spore  formation.  The  spores  formed 
after  fertilization  are  supplied  with  a  firm  protecting  covering,  and 
they  are  able  to  exist  outside  of  the  body  of  the  animal  in  which  they 
are  parasitic  as  mature  organisms.  The  spores  formed  asexually 
have  no  such  protecting  envelope,  and  cannot  live  outside  of  their 
host.  Since  the  spores  formed  under  such  different  circumstances 
differ  so  much  in  their  biologic  properties,  they  have  been  distinguished 
as  sporozoites,  those  formed  after  fertilization,  and  merozoites,  those 
formed  in  an  asexual  manner.  The  former,  can  carry  the  infection 
or  disease  from  one  host  to  another;  the  latter  can  only  carry  it 
from  one  part  of  the  infected  host  to  another,  but  they  cannot  enter 
the  outside  world. 

The  term  sporoblast  designates  the  mother  cell  in  which  the  sporo- 


536  GENERAL  CONSIDERATION  IN  PROTOZOA 

zoites  have  been  formed,  while  schizont  is  the  cell  which  gives  rise 
to  the  merozoites. 

Protozoa  not  Endowed  with  Eternal  Life. — It  was  formerly  believed 
that  the  unicellular  protozoa  could  continue  dividing  indefinitely, 
that  they  were  endowed  with  eternal  youth  and  eternal  life,  and  that 
they  did  not  go  through  a  period  of  maturity  and  still  less  through  a 
period  of  old  age,  followed  by  death.  It  was  first  shown  by  Biitschli, 
Hertwig  and  Maupas,  and  Calkins,  by  very  thorough  investigations, 
that  protozoa,  even  under  favorable  conditions,  after  a  certain 
number  of  generations  reach  a  condition  of  lowered  vitality  and 
depression.  In  consequence  of  this  they  are  no  longer  able  to  propa- 
gate and  die  unless  certain  changes  occur  which  lead  to  the  formation 
of  a  germ  plasm  which  permits  a  rejuvenation  by  sexual  reproduction. 
One  of  the  important  changes  indicating  maturity  and  the  necessity 
for  sexual  rejuvenation  and  reproduction  is  the  formation  of  the 
chromidia.  This  term,  as  explained,  designates  the  appearance  of 
chromatin  granules  derived  and  expelled  from  the  nucleus  into  the 
cytoplasm.  Schaudinn  has  shown  that  the  nuclei  of  conjugating 
gametes  are  developed  exclusively  from  such  extranuclear  chromatin. 
Mesnil,  therefore,  proposed  to  call  them  idiochromidia.  The  forma- 
tion of  the  extranuclear  chromidia  or  idiochromidia  in  protozoa  occurs 
by  nuclear  transfusion,  by  dissolution  of  nuclear  parts,  or  by  nuclear 
fragmentation. 

The  facts  as  to  maturity  and  senility  of  protozoa  show  that  these 
low  unicellular  animal  organisms  are  no  more  endowed  with  un- 
limited youth  and  unlimited  individual  life  than  the  higher  multi- 
cellular  animals  or  metazoa.  Both  in  their  body  possess  only  one 
substance  which  under  the  proper  conditions  of  sexual  union  is 
endowed  with  the  property  of  uninterrupted  propagation — that  is,  the 
germ  plasm. 

Metabolism  of  Protozoa. — Protozoa  can  in  general  only  live  where 
there  is  considerable  moisture.  They  may,  however,  in  the  encysted 
condition  and  under  other  circumstances,  withstand  drying  out  for 
a  shorter  or  longer  time,  and  then  be  like  the  spores  or  the  vegetative 
form  of  bacteria  under  similar  conditions,  in  a  state  of  suspended 
animation,  from  which  they  may  come  to  life  again  when  the  necessary 
amount  of  water  is  supplied.  There  are  a  few  protozoa,  which  like 
plants  possess  chlorophyl,  and  can  derive  their  food  and  build  up 
the  constituents  of  their  body  from  very  simple  compounds  forming 
carbohydrates  and  proteids  from  them.  However,  as  an  almost 
invariable  rule,  protozoa  cannot  subsist  on  such  simple  compounds, 
but  they,  like  the  zoometazoa,  require  carbohydrates  and  proteids  in 
order  to  supply  their  demand  for  growth  and  multiplication.  Most 
protozoa  are  supplied  with  organs  of  locomotion  particularly  for 
the  purpose  of  obtaining  food.  Such  organs  of  locomotion  act  by 
producing  in  a  fluid,  currents  which  bring  toward  the  primitive 
animal  organism  other  small  animal  organisms,  bacteria,  yeasts,  and 


CLASSIFICATION  537 

particles  of  plants  and  animals.  These  may  then  be  engulfed  through 
a  mouth  organ  or  they  may  simply  be  incorporated  into  the  protozoan 
protoplasm.  Some  of  the  latter,  like  ameba,  possess  as  organs  of 
locomotion  simple  protoplasmic  processes.  These  become  attached 
to  small  food  particles,  which  then  become  incorporated  into  the 
protoplasm  by  a  flowing  of  the  latter  around  the  material  intended 
for  food.  When  the  latter  are  incorporated  there  is  formed  in  the 
engulfing  protoplasm  a  food  vacuole  which  secretes  either  acids  or 
alkalies  and  a  digestive  ferment  of  the  peptic  or  tryptic  type.  From 
the  foodstuffs  certain  materials  are  extracted,  others  are  stored  as 
reserve  material  (particularly  oils  and  fats),  and  others  again  are 
expelled  as  waste  products.  An  exchange  of  gases  is  likewise  kept 
up  by  the  protozoa,  all  of  which  show  a  more  or  less  high  degree  of 
irritability  toward  chemical  and  physical  influences. 

Other  protozoa,  while  existing  under  the  same  general  laws  of 
metabolism,  have  by  parasitism  become  adapted  to  a  special  mode 
of  life.  They  exist  in  the  blood  serum  of  higher  animals  (trypano- 
somes)  or  they  invade  cells  of  the  host  (coccidia,  plasmodium  of 
malaria  of  man  and  birds),  and  they  then  generally  obtain  their 
food  supply  by  osmotic  processes.  While  the  organs  of  locomotion 
in  the  phylogenetic  development  of  the  protozoan  races  have  evidently 
been  formed  largely  with  the  object  of  securing  the  food  supply,  they 
have  also  been  used  extensively  as  the  basis  of  the  systematic  sub- 
division and  classification  of  the  species,  etc.,  of  the  phylum  protozoa. 

Classification. — The  classification  as  given  in  the  last  edition  of 
Calkins'  Protozoology  is  followed,  but  reference  is  made  only  to  those 
subdivisions  which  embrace  the  parasitic  and  pathogenic  protozoa 
more  fully  considered  in  the  following  pages. 

The  subphylum  sarcodina  is  defined  as  protozoa  showing  no 
connections  with  the  bacteria,  usually  of  simple  structure  and  char- 
acterized mainly  by  motile  organs  in  the  form  of  changeable  proto- 
plasmic processes,  the  pseudopodia.  In  this  subphylum  the  subclass 
ameba  is  of  particular  interest.  It  includes  the  more  common 
forms  of  rhizopods,  with  blunt  or  lobose  pseudopodia,  which  do  not 
anastomose  on  touching  one  another.  The  protoplasmic  body  may 
or  may  not  possess  a  shell. 

The  subphylum  mastigophora  comprises  protozoa  in  which  the 
kinoplasm  is  concentrated  in  the  form  of  one  or  more  vibratile  or 
undulating  motile  processes  called  flagella,  or  in  a  kinetonucleus 
which  may  lie  inside  or  outside  of  the  trophonucleus.  This  subphylum 
comprises  the  very  important  flagellates,  trypanosoma  and  herpeto- 
monas  and  the  less  important  cercomonas  and  trichomonas. 

The  subphylum  infusoria  includes  the  protozoa  in  which  the 
motor  apparatus  is  in  the  form  of  cilia,  either  simple  or  united  into 
membranes,  membranelles  or  cirri.  The  cilia  may  be  permanent  or 
limited  to  the  young  stages.  With  a  micronucleus  and  a  macro- 
nucleus,  reproduction  is  effected  by  simple  transverse  division  or 


538  GENERAL  CONSIDERATION  OF  PROTOZOA 

budding.  This  subphylum  is  of  little  importance  from  the  standpoint 
of  the  pathologist.  One  species  only  must  be  considered,  namely 
Balantidium  coli,  and  this  is  of  doubtful  pathogenic  importance. 

The  subphylum  sporozoa  comprises  parasitic  protozoa  without 
motile  organs,  but  which  are  capable  of  moving  from  place  to  place 
by  structural  modification  of  one  kind  or  other.  This  subphylum  is, 
again,  of  great  importance,  because  it  includes  pathogenic  coccidia, 
the  malarial  organisms  of  man  and  birds,  and  the  piroplasma  which 
cause  Texas  fever  in  cattle  and  other  animal  piroplasmoses. 


QUESTIONS 

1.  Give  a  definition  of  the  phylum  protozoa. 

2.  Where  do  they  occur? 

3.  What  is  their  general  morphology? 

4.  Compare  the  individual  variability  in  bacteria  and  protozoa. 

5.  Define  the  terms  spongioplasm,  hyaloplasm,  ectoplasm,  entoplasm. 

6.  What  are  plastids  and  chromatophores  ? 

7.  Define  the  terms  macronucleus,  micronucleus,  trophonucleus,  karyogonad, 
chromatin,  kinoplasm,  archoplasm. 

8.  What  are  the  various  organs  of  locomotion  found  in  protozoa? 

9.  Define  the  terms  lobopodia,  filopodia,  actinopodia,  cilia. 

10.  Describe  the  simplest  form  of  reproduction  in  protozoa. 

11.  What  is  meant  by  mitotic,  what  by  amitotic  nuclear  division? 

12.  What  is  karyomitosis  ? 

13.  What  are  chromosomes?     What  centrosomes?     What  the   achromatic 
spindle  ? 

14.  What  is  a  blepharoblast  ? 

15.  What  is  autogamy  or  automyxis? 

16.  What  are  chromidia  or  idiochromidia  ? 

17.  What  is  endogamy? 

18.  What  is  exogamy? 

19.  What  are  gametes? 

20.  What  is  isogamy,  what  anisogamy? 

21.  What  are  sporozoites  ?    What  merozoites? 

22.  What  is  a  sporoblast,  what  a  schizont? 

23.  Name  the  different  forms  of  reproduction  which  occur  among  protozoa. 

24.  Discuss  the  metabolism  of  protozoa.    Can  they  ever  use  simple  compounds 
as  food  ? 

25.  Discuss  organs  of  locomotion  with  reference  to  food  supply. 

26.  Discuss  intracellular  digestion  by  protozoa. 

27.  Give  the  characteristics  of  the  subphylum  sarcodina  and  name  some 
pathogenic  microorganisms  belonging  to  this  subphylum. 

28.  Give  the  same  with  reference  to  the  subphylum  mastigophora. 

29.  Give  the  same  with  reference  to  the  subphylum  infusoria. 

30.  Give  the  same  with  reference  to  the  subphylum  sporozoa. 


CHAPTEK    LI. 

CLASSIFICATION  AND  MORPHOLOGY  OF  AMEB A— CULTIVATION- 
PATHOGENIC  AMEBA— ENTERO-HEPATITIS  IN 
TURKEYS— BALANTIDIUM   COLI. 

AMEBA. 

Morphology. — Ameba  is  a  genus  of  protozoa  belonging  to  the  class 
of  rhizopoda  of  the  subphylum  sarcodina.  The  name  is  derived 
from  a  Greek  word  which  means  change,  indicative  of  the  fact  that 
amebse  are  protozoan  organisms  which,  under  favorable  conditions, 
constantly  change  their  form.  This  is  brought  about  by  currents  in 
their  protoplasm  and  by  the  formation  of  processes  extending  from 
the  periphery.  These  processes,  which  are  called  pseudopodia  (false 
feet),  serve  as  organs  of  locomotion.  If  an  ameba  is  suspended  in  a 
fluid  it  is  likely  to  send  out  short  pseudopodia  in  every  direction.  As 
soon  as  one  pseudopodium  touches  a  small  solid  particle  the  other 
pseudopodia  are  drawn  in  and  the  remaining  one  elongates  and  draws 
the  whole  organism  toward  the  particle  to  which  it  has  fastened  itself. 
If  amebse  are  studied  on  a  slide  under  the  microscope  it  can  be 
seen  how  they  always  draw  their  body  along  on  a  pseudopodium 
extending  out  from  the  periphery.  The  motion  is  a  very  peculiar  one. 
It  is  really  not  so  much  a  drawing  of  the  protoplasm  as  a  flowing 
of  the  latter  in  the  direction  of  the  outstretched  pseudopodium,  which 
is  generally  lobose  or  lobular  in  shape.  Amebse  generally  exhibit 
a  round  or  oval,  more  or  less  vesicular  nucleus.  The  chromatin  is 
distributed  along  the  periphery  of  the  nucleus  and  the  interior  shows 
a  central  granule.  The  protoplasm  of  amebse  is  generally  more  or 
less  finely  granular  and  very  frequently  contains  one  or  more  con- 
tractile vacuoles  which  preferably  empty  their  fluid  toward  the  outside. 
The  cytoplasm  is  generally  differentiated  into  an  entoplasm,  and  an 
often  strongly  hyalin,  ectoplasm.  Multiplication  of  amebse  occurs 
either  by  fission  with  an  amitotic  division  of  the  nucleus,  or  the 
ameba  may  become  encysted,  forming  in  its  interior  a  number  of 
young  amebse  which,  after  rupture  of  the  cyst  membrane,  are  set 
free  and  grow  rapidly.  Spore  formation  has  also  been  observed. 
This  is  preceded  by  the  expulsion  of  chromidia  or  idiochromidia 
from  the  nucleus  into  the  cytoplasm.  Small  masses  of  chromatin 
reach  the  periphery,  are  extruded  from  it,  and  finally  cut  off  with  a 
small  amount  of  protoplasm.  From  these  spores  young  amebse  are 
developed.  Amebse  are  found  in  the  outside  world  and  in  moist 
soil,  where  they  lead  a  purely  saprophytic  existence,  or  they  may  be 


540         CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 

found  in  the  intestines  of  a  great  variety  of  animals,  where  they  lead 
a  parasitic,  but  as  a  rule  perfectly  harmless,  life. 

Microscopic  Study. — The  study  of  ameba  is  to  be  undertaken  on 
fresh  preparations  in  the  live  state,  and  it  is  then  best  to  use  the  fluid 
in  which  they  naturally  occur.  Saprophytic  amebae  are,  therefore, 
best  studied  in  the  water  in  which  they  are  found;  parasitic  amebse 
can  be  studied  in  feces  or  in  scrapings  from  the  intestines,  perhaps 
mixed  with  a  small  amount  of  physiologic  salt  solution.  Of  such 
fluids  containing  saprophytic  or  parasitic  amebse  a  hanging  drop  may 
be  made  or  a  drop  of  the  fluid  is  placed  on  a  slide  and  covered  with 
a  cover-glass.  If  the  cover-glass  be  used  it  is  well  to  supply  it  with 
four  very  short  wax  feet,  so  that  the  weight  of  the  cover  does  not 
compress  the  amebse,  as  they  are  much  more  sensitive  to  insults 
than  bacteria,  and  a  small  degree  of  pressure  may  injure  them.  When 
amebse  in  semisolid  or  semifluid  feces  are  studied  it  is  not  necessary 
to  supply  the  cover-glass  with  wax  feet,  because  such  material  generally 
contains  a  sufficient  number  of  undigested  particles  to  furnish  a 
support  for  the  cover-slip.  When  parasitic  intestinal  amebse  are 
studied  in  the  fresh  state  it  is  well  to  warm  the  slide  and  cover-glass, 
because  such  amebse  lose  their  motility  as  soon  as  they  are  materially 
cooled.  Care  should  be  taken  not  to  heat  the  slide  and  cover-glass 
too  much,  as  otherwise  the  amebse  go  into  a  condition  of  "heat  rigor," 
and  become  likewise  immobile.  The  best  method  of  warming  the 
slide  and  cover-slip  properly  is  to  immerse  them  for  some  time  in 
water  a  little  warmer  than  body  temperature  (about  40°  C.). 

Staining  Properties. — Amebse  should  also  be  studied  in  stained 
preparations.  The  simplest  method  of  studying  stained  amebse 
consists  in  the  preparation  of  thin  smears  or  spreads  on  cover-glasses. 
For  the  study  of  intestinal  amebae,  Craig  recommends  Oliver's 
modification  of  Wright's  staining  method.  The  spread  is  made  on 
the  slide  and  is  allowed  to  become  air  dry,  and  then  a  few  drops  of 
Wright's  stain  are  poured  on  the  slide.  The  stain,  being  dissolved 
in  methyl  alcohol,  fixes  at  the  same  time  that  it  dyes.  The  stain 
is  allowed  to  act  for  five  minutes,  then  enough  distilled  water  is 
added  to  cause  a  slight  metallic  scum  to  appear  on  the  surface.  The 
dilute  stain  then  remains  on  the  slide  for  ten  minutes  longer  and 
the  preparation  is  finally  well  washed  in  distilled  water  and  dried 
between  filter  paper.  The  slide  is  not  mounted  in  Canada  balsam, 
but  is  examined  directly  with  the  oil-immersion  lens.  If  after  exam- 
ination the  specimen  is  to  be  preserved  the  immersion  oil  is  washed  off 
with  xylol.  Walker  recommends  the  chloride  of  iron  hematoxylin 
method  of  Mallory  for  staining  amebse  in  slide  and  cover-glass 
preparations.  The  smears  after  being  air  dry  must  first  be  fixed 
a  short  time  in  Zenker's  solution,1  then  washed  successively  in  water, 

1  Zenker's  solution  is  composed  of  bichloride  of  mercury,  5  grams;  bichromate  of  potash, 
2.5  grams;  sulphate  of  sodium,  1  gram;  glacial  acetic  acid,  5  c.c.;  and  water  enough  to  make 
100  c.c. 


CULTURAL  CHARACTERISTICS  541 

iodine-alcohol,  and  pure  alcohol,  and  dried.     The  steps  of  the  iron 
hematoxylin  method  are  the  following : 

1.  Stain  smears  for  three  to  five  minutes  in  a  10  per  cent,  watery 
solution  of  ferric  chloride. 

2.  Drain  and  blot  the  cover-glass,  then  pour  over  it  a  few  drops  of 
a  freshly  prepared  1  per  cent,  aqueous  solution  of  hematoxylin.     If 
all  the  hematoxylin  is  precipitated  by  the  excess  of  ferric  chloride, 
pour  off  the  solution  and  add  a  fresh  supply.     In  three  to  five  minutes 
the  sections  will  be  colored  a  dark  bluish  black. 

3.  Wash  in  water. 

4.  Decolorize  and  differentiate  in  a  J  per  cent,  aqueous  solution 
of  ferric  chloride.    The  cover-glass  must  be  kept  constantly  moving 
in  the  solution.    The  differentiation  will  be  complete  in  a  few  seconds 
to  several  minutes. 

5.  Wash  in  water,  dry,  and  examine. 

If  the  differentiation  is  not  sufficient  the  preparation  must  again 
be  washed  in  the  \  per  cent,  solution  of  ferric  chloride.  Mallory 
states  that  the  principal  point  in  this  method  is  first  to  stain  very  deeply 
and  then  to  differentiate  properly.  The  nuclei  of  amebse  stain  sharply 
with  this  method. 

Cultural  Characteristics. — Attempts  to  cultivate  amebse  had  been 
made  for  a  number  of  years,  but  not  much  progress  was  made  until 
Musgrave  and  Clegg  devised  a  method  which  is  now  generally  used, 
either  according  to  the  original  recommendation  or  with  some  slight 
modification.  The  principle  of  this  method  consists  in  preparing  a 
culture  soil,  comparatively  poor  for  bacteria,  which  will  enable  them 
to  thrive  only  moderately,  but  sufficiently  to  serve  as  food  for  the 
amebse,  which  require  proteids  for  their  metabolism  and  cannot 
utilize  simple  compounds  like  plants.  Musgrave  and  Clegg's  medium 
consists  of: 

Agar  .      .  '    .  '   . .      .          20  gr. 

Sodium  chloride  .      .     -. .  0 . 3  to  0 . 5  gr. 

Extract  of  beef    .      .      .      .      ..     .    .  ,      .      .      .      .      ...      .     ,  0.3to0.5gr. 

Water.      .      .....      .      .      .      .      ,  '   .      .      .      .      .      .  .      .      1000  c.c. 

This  is  prepared  in  the  same  manner  as  ordinary  agar  and  made  1  per 
cent,  alkaline  to  phenolphthalein.  The  medium  is  sterilized  in  tubes 
and  from  these  Petri  dishes  are  filled  in  the  ordinary  manner,  but, 
of  course,  without  inoculating  anything  into  the  sterile,  melted  agar 
before  it  is  poured  out  into  the  lower  pla'te  of  the  Petri  dish.  The  agar 
is  there  allowed  to  solidify.  If  amebse  are  to  be  cultivated  from  water 
or  from  water  mixed  with  vegetable  material,  100  to  500  c.c.  of  the 
fluid  are  placed  in  a  flask  and  0.5  to  1  c.c.  of  ordinary  nutrient  bouillon 
is  added  for  each  100  c.c.  of  amebse-containing  fluid.  The  flasks 
are  then  set  aside  and  kept  at  room  temperature  for  from  twenty-four 
to  seventy-two  hours,  when  a  few  loopful  of  fluid  may  be  removed 
from  the  surface,  spread  on  a  slide  and  examined  fresh  for  the  presence 


542  CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA' 

of  amebse.  If  microscopic  examination  reveals  amebse,  one  or  more 
loopfuls  of  the  surface  fluid  containing  them  is  streaked  over  the 
surface  of  the  set  agar  in  the  Petri  dishes.  In  the  course  of  from 
six  to  forty-eight  hours  the  plates  are  to  be  examined  under  a  low 
power  of  the  microscope  in  the  same  manner  as  for  bacterial  colonies. 
If  the  amebse  have  multiplied,  at  they  usually  do,  they  can  be  recog- 
nized under  the  low  power  of  the  microscope  as  highly  refractive 
bodies.  From  such  plates  others  can  be  inoculated  by  making  trans- 
plants and  again  streaking  the  agar  on  the  surface.  In  trying  to  obtain 
a  growth  of  intestinal  amebse  it  is  necessary  to  streak  small  particles 
of  feces,  best  some  mucous  flocculi  picked  up  with  the  platinum  loop 
over  agar  plates,  because  such  parasitic  amebse  do  not  multiply  as 
easily  in  fluid  as  do  saprophytic  amebse  from  ordinary  outside  sources. 

FIG.  184  FIG.  185 


Ameba  from  a  case  of  tropical  dysentery  Encysted  forms  of  an  ameba  from  an  old 

in  man.    Twelve  hours'  culture.     (Musgrave  culture.     (Musgrave  and  Clegg.) 

and  Clegg.) 

When  amebse  grow  after  such  an  inoculation  several  kinds  may  be 
present  in  addition  to  a  variety  of  bacteria.  It  is,  however,  necessary 
to  isolate  one  kind  of  ameba  and  to  cultivate  it  with  one  known  kind 
of  bacterium.  No  one  has  ever  succeeded  in  cultivating  amebse  alone 
in  absolutely  pure  culture,  because  they  evidently  need  for  their 
nutrition  live,  unchanged  proteid  material.  The  best  that  has  so 
far  been  accomplished  has  been  to  obtain  amebse  in  symbiotic 
community  with  one  known  species  of  bacterium.  Such  a  culture 
has  been  called  by  Frosch  "a  mixed  pure  culture  of  amebce."  Mus- 
grave and  Clegg  succeeded  in  getting  such  pure  cultures  in  a  manner 
described  by  them  as  follows : 

"Select  a  plate  culture  on  which  the  parasites  are  well  distributed 
and  after  removing  the  cover,  place  the  plate  with  the  open  side  up 
on  the  stage  of  the  microscope.  By  searching  the  edges  of  the  growth 


CULTURAL  CHARACTERISTICS  543 

with  a  low-power  objective,  places  will  be  found  where  the  ameba 
are  some  distance  apart.  After  locating  a  satisfactory  parasite, 
which  should  be  one  on  the  surface  of  the  medium,  as  practically  all 
of  them  are,  and  having  determined  that  there  are  no  others  in  the 
field,  either  on  the  surface  or  at  a  depth,  swing  a  perfectly  dry  and 
clean  high-power  lens  in  place  and  gently  lower  it  until  the  entire 
surface  is  in  contact  with  the  medium.  Raise  the  lens  quickly,  swing 
in  the  low-power  objective  and  determine  whether  the  ameba  is 
still  present  or  has  been  picked  up.  If  it  has  been  picked  up,  which 
after  some  practice  may  be  done  two  or  three  times  out  of  five,  the 
lens  to  which  the  ameba  adheres  is  removed,  and,  by  gently  rubbing 
its  surface  over  that  of  a  plate  containing  the  hardened  medium  the 
organism  may  be  transferred.  In  this  manner  a  pure  culture,  so 
far  as  amebae  are  concerned,  may  be  obtained.  That  only  one 
ameba  has  been  carried  over  by  this  method  may  still  farther  be 
verified  by  examining  with  a  low-power  objective  the  closed  inverted 
plate  on  which  it  has  been  inoculated.  Another  useful  result  of  a 
careful  application  of  this  method  is  the  aid  it  gives  in  obtaining  pure 
cultures  of  an  ameba  and  of  a  single  bacterium.  The  lens,  of  course, 
picks  up  the  bacteria  from  a  small  field  immediately  surrounding 
the  ameba;  and  as  such  isolated  ameba  is  often  surrounded  by  one 
kind  of  bacterium  only,  with  the  aid  of  a  careful  bacteriologic  tech- 
nique the  pure  cultures  desired  may  sometimes  be  obtained  in  this 
manner."  Musgrave  and  Clegg  have  used  the  preceding  method  in 
most  cases  and  have  found  it  satisfactory.  It  would  be  possible  to 
obtain  the  desired  results  more  easily  and  with  greater  constancy 
by  means  of  Unna's  bacterial  harpoon  or  a  specially  constructed 
lens,  with  a  short  adjustable  focus  and  a  cup-shaped  extremity,  like 
the  marking  arrangement  which  has  been  suggested  for  locating 
special  fields  in  permanent  preparations. 

Parasitic  amebse  obtained  in  cultures  on  plates  growing  there  in 
symbiosis  with  bacteria  will  not  grow  with  any  and  all  bacteria, 
but  they  are  quite  selective,  at  least,  at  first,  and  it  is,  therefore, 
necessary  to  obtain  from  the  mixed  culture  on  the  first  plates  all 
bacteria  present  in  pure  cultures.  After  these  have  been  obtained 
and  a  number  of  individual  amebse  have  been  picked  out  by  the 
method  described  above  and  transferred  to  fresh  plates  such  planted 
amebse  are  surrounded  by  several  concentric  streaks  of  bacteria 
obtained  from  a  pure  culture.  If  the  necessary  precautions  have  been 
taken,  most  amebse,  as  they  multiply,  will  quite  generally  spread 
rapidly  over  the  plate,  and  in  passing  through  the  rings  of  growing 
bacteria  they  will  lose  the  organisms  with  which  they  started  and 
take  up  those  forming  the  rings.  In  from  twenty-four  to  seventy- two 
hours  the  protozoa  will  have  passed  one  or  more  of  the  rings,  and 
from  such  locations  they  may  be  taken  for  transplanting.  It  sometimes 
happens  that  they  appear  on  the  first  plate  in  pure  cultures  with  the 
desired  organism,  but  usually  one  or  more  transplants  to  the  same 


544         CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 

medium  are  necessary  before  this  end  is  reached.  The  further 
inoculations  are  made  with  amebae  obtained  from  outside  the  largest 
ring  on  the  next  preceding  culture. 

This  method  is  simple  in  execution,  and  the  entire  process  may 
be  watched  under  the  microscope  by  inverting  the  plate  and  using  a 
low  power,  according  to  the  method  employed  in  studying  colonies  of 
bacteria.  With  a  low  power  the  wanderings  of  the  amebae  and  even 
their  multiplication  can  be  kept  under  observation. 

The  ring-shaped  smear  of  bacteria  has  several  advantages  over 
one  covering  the  entire  surface  of  the  plate.  In  the  first  place  amebae 
develop  more  rapidly  by  its  use,  and  secondly,  they  lose  the  original 
organisms  much  more  rapidly  than  when  moving  constantly  over  a 
bacterial  substratum. 

Another  feature  which  commends  this  method  is  the  facility  with 
which  it  lends  itself  to  a  determination  of  the  symbiotic  value  of  a 
given  organism.  If  such  an  organism  for  any  reason  is  not  satisfactory 
to  the  amebae  they  will  not  mix  with  or  cross  the  bacterial  rings. 
In  some  instances,  where  the  organism  is  particularly  unfavorable, 
the  amebae,  after  wandering  up  to  the  inner  margin  of  the  first 
ring,  encyst,  and  no  further  progress  is  made.  On  the  other  hand, 
where  the  antipathy  is  less  marked,  the  progress  is  simply  delayed 
until  the  bacteria  carried  over  in  inoculating  the  amebae  have  mixed 
with  or  crossed  the  ring,  whereupon  the  amebae  follow  them. 

When  amebae  have  been  isolated  and  grown  in  pure  culture  with 
a  satisfactory  symbiotic  organism  it  is  sometimes  difficult  to  transfer 
them  to  another.  This  is  best  overcome  by  first  cultivating  the  pro- 
tozoa for  a  short  time  on  a  mixed  culture  of  the  two  organisms  and 
then  isolating  them  with  the  desired  one  by  the  use  of  the  method 
described.  Even  by  this  means  success  is  often  doubtful  and  some- 
times impossible  of  attainment. 

Among  the  bacteria  with  which  amebae  have  entered  into  symbiotic 
community  as  reported  by  various  observers  are  the  spirillum  of 
Asiatic  cholera,  typhoid  bacillus,  Bacillus  coli,  Bacilli  fluorescens 
liquefaciens  and  non-liquefaciens,  Staphylococcus  pyogenes  aureus, 
Bacillus  pyocyaneus,  Bacillus  ruber,  spirillum  of  Metchnikoff,  various 
other  bacteria,  and  also  yeast  cells. 

Walker,  starting  out  from  pure  mixed  cultures,  according  to  the 
above  method,  has  modified  it  in  such  a  manner  that  he  could  study 
the  whole  development  under  the  microscope.  He  calls  his  device 
the  "hanging-plate  culture,"  and  prepares  it  as  follows:  A  thin  |  inch 
cover-glass  is  flamed  and  placed  under  a  flamed  watch-glass.  With 
a  large  platinum  loop  a  large  loopful  of  melted  sterile  agar  is  trans- 
ferred to  the  sterile  cover  and  spread  in  a  uniform,  thin,  and  circum- 
scribed layer.  This  film  of  agar  will  solidify  almost  instantly,  and  it 
is  at  once  inoculated  from  a  pure  mixed  ameba  culture.  The  cover- 
glass  culture  is  then  inverted  over  a  concave  slide  which  has  been 
flamed,  cooled,  and  rimmed  with  vaselin.  On  such  cover-glasses 


ENTAMCEBA  COLI  545 

amebae  multiply  as  freely  as  on  Petri  dishes.  The  film  of  agar  medium 
is  thin  enough  to  permit  the  use  of  a  2  mm.  oil-immersion  lens. 

Pathogenic  Amebae. — Amebae  have  frequently  been  found  in  the 
intestinal  tract  of  man  and  the  lower  animals.  They  were  perhaps 
first  seen  in  1859  by  Lamble  and  demonstrated  beyond  a  doubt 
by  Loesch  in  1875,  who  found  them  in  the  discharges  of  a  patient 
suffering  from  chronic  dysentery.  Loesch  called  the  organism 
Amoeba  coli,  and  claimed  that  he  was  able  to  produce  dysentery  in 
dogs  by  the  injection  of  the  feces  containing  them.  Amebse  as  the 
possible  cause  of  disease  in  man  or  animals,  however,  did  not  attract 
much  attention  until  Robert  Koch,  while  studying  Asiatic  cholera 
in  Egypt,  found  them  in  the  tissues  of  the  intestines  of  three  persons 
who  had  died  from  chronic  dysentery.  Koch's  observations  stimu- 
lated the  work  of  Kartulis,  who  found  amebse  in  the  stools  of  150 
sufferers  from  dysentery,  and  who  published  his  investigations  in 
1886.  Observations  in  this  country  were  then  made  by  Osier, 
Musser,  Stengel,  Dock,  Councilman  and  Lafleur,  Harris  and  others. 
While,  for  a  number  of  years,  there  has  been  no  doubt  that  amebse 
are  found  in  certain  dysenteries  in  man,  their  role  in  the  production 
of  this  disease  has  been  again  and  again  in  doubt,  as  it  has  been 
shown  that  they  occur  in  all  forms  of  diarrhea  and  frequently  in 
the  stools  of  perfectly  healthy  persons  and  in  the  intestines  of  many 
species  of  animals  which  are  in  a  normal  condition  of  health.  Walker 
recently  described  37  species  of  amebse  (including  several  previously 
undescribed  species)  in  the  intestines  of  man,  the  horse,  pig,  dog, 
cat,  rabbit,  guinea-pig,  rat,  house  mouse,  white  mouse,  etc.,  the 
great  majority  of  which,  beyond  doubt,  are  perfectly  harmless  com- 
mensales  in  the  intestines  of  their  host.  The  question  of  the  patho- 
genicity  of  amebse  in  man  has  been  much  clouded  because  most 
observers  had  not  learned  to  differentiate  a  harmless  commensale 
from  a  truly  pathogenic  organism.  Two  observers  in  particular, 
Schaudinn  and  Craig,  however,  have  clearly  shown  that  one  common 
non-pathogenic  ameba  and  one  pathogenic  species  are  really  found 
in  the  intestines  of  man.  Schaudinn  was  the  first  to  describe  these 
two  types  definitely.  Craig  made  some  early  observations  independent 
of  Schaudinn,  and  later  confirmed  all  of  Schaudinn's  observations  as 
to  the  fundamental  difference  between  the  two  types.  Schaudinn 
named  the  harmless  commensale  in  the  intestines  or  man  Entamceba 
coli,  and  the  pathogenic  organism  which  is  the  cause  of  so-called 
amebic  dysentery  Entamosba  hystolytica  (histolytica,  tissue  dissolving). 

Entamoeba  Coli. — This  harmless  commensale  was  found  by  Schaudinn 
in  the  feces  obtained  after  purging  in  50  per  cent,  of  healthy  persons 
examined  in  West  Prussia;  in  20  per  cent,  of  persons  examined  in 
Berlin,  and  in  66  per  cent,  of  persons  examined  along  the  shores  of 
the  Adriatic.  Craig  found  this  organism  in  65  per  cent,  of  healthy 
American  soldiers  examined  in  San  Francisco;  Ashburn  and  Craig, 
in  71  per  cent,  of  healthy  American  soldiers  in  Manila,  and  Vedder, 
35 


546         CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 

in  50  per  cent,  of  American  soldiers  in  Manila  and  72  per  cent,  of 
Philippine  scouts.  In  order  to  find  Amoeba  coli  in  the  stools  of 
healthy  persons  it  is  practically  always  necessary  to  administer  a 
large  dose  of  salts,  such  as  sulphate  of  magnesium  or  sodium.  The 
discussion  as  to  pathogenic  and  non-pathogenic  amebse  has  been  of 
the  greatest  importance,  since  it  makes  a  good  deal  of  difference 
whether  all  amebse  may  occasionally  become  pathogenic,  or  whether 
among  them,  as  among  bacteria,  certain  definite  types  produce  specific 
diseases,  while  others  are  perfectly  harmless  and  non-pathogenic. 
In  animals  the  same  conditions,  of  course,  prevail,  and  in  them, 
apparently,  as  in  man,  most  amebse  inhabit  the  intestines  as  harmless 
commensales,  but  likewise  one  species  has  already  been  found  which 
evidently  is  a  very  dangerous  pathogenic  parasite. 

Entamceba  coli,  as  described  by  Schaudinn,  Craig,  and  others,  con- 
sists of  a  mass  of  protoplasm,  containing  a  well-defined  nucleus  and 
generally  one  or  more  nucleoli.  Sometimes  a  non-contractile  vacuole  is 
present,  but  rarely,  if  ever,  more  than  one  vacuole.  The  differ- 
entiation between  the  entoplasm  and  ectoplasm  is  very  faint;  the 
organism  is  very  sluggishly  motile.  Reproduction  under  favorable 
conditions  occurs  by  simple  division,  under  unfavorable  conditions 
after  encystation  followed  by  the  formation  of  eight  daughter  cells 
in  the  cyst.  The  daughter  cells  after  solution  or  rupture  of  the  cyst 
membrane  are  set  free  and  develop  into  young  amebse.  Entamoeba 
coli  varies  in  size  between  8  to  50  micra,  generally  between  25  to  30 
micra.  When  not  in  motion  it  is  spherical  in  shape.  Its  pseudopodia 
are  rounded  or  lobose,  never  sharply  pointed.  It  is  of  a  dull  grayish 
color,  and  takes  up  red  blood  corpuscles  very  rarely  even  if  they  are 
present  in  the  feces.  Bacteria  are  often  found  in  the  finely  granular 
protoplasm.  The  nucleus,  5  to  8  micra  in  diameter,  is  generally 
situated  a  little  to  one  side  of  the  centre.  It  has  a  thick,  easily  seen 
nuclear  membrane  and  possesses  a  large  amount  of  chromatin.  In 
stained  specimens  the  ectoplasm  dyes  very  dimly,  the  entoplasm 
intensely.  The  latter  is  composed  of  well-defined  granules,  among 
which  engulfed  bacteria  can  generally  be  seen;  the  chromatin  of  the 
nucleus  is  shown  as  short  strands  or  round  granules.  The  encysted 
forms  do  not  take  the  stain  on  account  of  their  firm  capsule.  When 
encystation  occurs  the  organism*  becomes  perfectly  motionless  and 
develops  from  its  spherical  periphery  a  highly  refractive  hyaline 
membrane  which  finally  acquires  a  double  outline  or  contour  and 
appears  irregularly  striated.  During  the  formation  of  the  cyst  wall 
the  organism  apparently  contracts  and  loses  about  one-third  of  the 
diameter  it  had  when  encystation  began.  The  protoplasm  in  the 
cyst  becomes  hyalin,  the  nucleus  breaks  up,  and  eight  daughter  nuclei 
are  formed,  around  these  cytoplasm  is  distributed,  and  in  this  manner 
eight  young  cells  originate  in  the  interior  of  the  cyst. 

Entamoeba  Hystolytica. — This  is  the  pathogenic  type  of  ameba 
parasitic  in  the  intestines  of  man,  and  is  the  cause  of  chronic  amebic 


ENTAMOEBA  HYSTOLYTICA 


547 


dysentery.  It  penetrates  into  the  intestinal  mucosa  and  submucosa, 
and  in  this  manner  produces  ulceration.  It  can  even  be  carried  to 
the  liver  and  there  produce  abscess.  Amebic  dysentery  is  most 
prevalent  in  the  tropics,  but  it  is  also  found  in  the  United  States, 
particularly  in  the  Southern  States.  The  organism  consists  of  a  mass 
of  protoplasm,  contains  a  nucleus,  and  generally  several  non-contractile 
vacuoles.  It  is  round  when  at  rest.  It  is  generally  larger  than 
Entamoeba  coli.  Craig  gives  its  average  diameter  as  35  micra,  and 
says  that  he  has  seen  individuals  as  large  as  60  to  70  micra.  In 
full-grown  individuals  the  differentiation  into  a  granular  entoplasm 
and  a  hyaline  ectoplasm  is  very  marked.  The  latter  forms  a  consider- 

FIG.  186 


Entamoeba  hystolytica.  (After  Craig.)  A,  organism  showing  rods  and  granules  of  chro- 
matin  in  the  nucleus,  vacuole  with  some  stained  substance,  and  dense  ectoplasm;  B,  the  chro- 
matin  of  the  nucleus  passing  into  the  cell  plasm,  where  it  is  distributed  as  chromidia,  shown 
in  C;  D,  aggregation  of  chromidia  to  form  secondary  nuclei;  E,  "spore  formation"  by  budding; 
F,  spores  of  Entamceba  histolytica  as  seen  in  feces. 

able  portion  of  the  entire  cytoplasm;  it  is  highly  refractive  and  glass- 
like  in  appearance.  It  can  generally  be  seen  at  its  best  when  the 
organisms  are  examined  in  a  warm,  fresh  stool.  Here  it  is  generally 
very  lively  motile,  much  more  so  than  Entamoeba  coli.  When  pseudo- 
podia  are  formed  a  rapid  or  also  more  slow  outflow  of  the  hyaline  ecto- 
plasm takes  place,  and  the  pseudopodium  is  first  formed  of  it  alone, 
later  the  granular  entoplasm  also  flows  in.  Schaudinn  and  Juergens 
believe  that  the  power  of  the  Entamreba  hystolytica  to  penetrate  into 
tissues  depends  primarily  upon  the  evidently  very  firm  tenacious 
ectoplasm.  The  nucleus  is  not  easily  distinguishable  in  the  live 
unstained  condition,  and  it  contains  a  small  amount  of  chromatin 
only.  The  pathogenic  entameba  when  found  in  bloody  stools  often 


548          CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 

contains  several  and  sometimes  many  red  blood  corpuscles.  These 
are  digested  in  the  interior  of  the  parasite,  and  its  protoplasm  and 
the  fluid  in  the  vacuoles  frequently  show  a  slight  greenish  tinge.  If 
stained  with  Wright's  stain  the  ectoplasm  stains  very  intensely,  while 
the  entoplasm  stains  lightly.  Entamoeba  coli  behaves  in  exactly  an 
opposite  manner.  The  nucleus  of  Entamoeba  hystolytica  stains  poorly 
on  account  of  the  scanty  amount  of  chromatin.  Reproduction  occurs 
by  division  and  budding  with  spore  formation.  The  latter  appears 
to  occur  in  the  intestines  of  man  when  conditions  become  unfavorable 
to  the  organism  and  when  it  becomes  advantageous  for  it  to  assume 
the  more  resistant  spore  form.  Then  the  nucleus  expels  and  dis- 
tributes most  of  its  chromatin  into  the  cytoplasm,  the  expelled  chrom- 
atin collects  into  small  masses,  and  these  reach  the  ectoplasm,  where 
they  become  protruded  with  some  cytoplasm,  beyond  the  periphery 
of  the  main  body,  and  are  finally  cut  off  from  the  mother  cell  entirely. 

Human  feces  containing  Entamoeba  hystolytica,  when  injected 
into  the  rectum  of  kittens,  produce  typical,  generally  fatal  attacks  of 
amebic  dysentery.  Upon  postmortem  examination  the  character- 
istic ulcerative  changes  of  the  disease  are  found  in  the  intestines. 

From  a  study  of  various  cultures  of  amebse,  Walker  came  to  the 
conclusion  that  Schaudinn's  observation  on  the  differences  between 
Entamoeba  coli  and  Entamoeba  hystolytica  were  incorrect;  but,  as 
Craig  properly  remarks,  Walker  studied  only  a  very  few  cases  of 
human  dysentery,  and  confined  his  observations  to  a  few  cultures  of 
intestinal  amebse  from  human  sources.  The  characteristic  re- 
production by  budding  in  Entamoeba  hystolytica,  however,  cannot 
be  observed  in  artificial  cultures,  but  must  be  studied  under  the  natural 
conditions  in  which  this  pathogenic  organism  it  found  in  the  intestines 
and  discharges  of  man. 

New  Species  of  Pathogenic  Amebse.— -Several  new  species  of  amebse 
pathogenic  to  man  have  recently  been  described  by  observers  working 
on  cases  of  chronic  tropical  dysentery  in  different  parts  of  the  world. 

Viereck  studied  the  stools  of  62  cases  of  dysentery  from  Africa, 
Europe,  and  South  America.  He  found  living  amebse  Jn  37  cases, 
and  bodies  which  he  thought  were  amebse  in  17  cases.  In  2  cases 
he  encountered  an  ameba  which  resembled  Entamoeba  coli  more 
than  it  did  Entamoeba  hystolytica,  but  it  formed  four  cysts  instead 
of  eight.  It  produced  dysentery  in  cats.  Viereck  also  found  this 
ameba  in  non-dysenteric  stools,  and  suggested  that  it  might  be  a 
variety  of  Entamoeba  coli.  He  called  it  Entawceba  tetragena. 

Hartmann,  almost  simultaneously  with  Viereck,  described  an 
ameba  which  was  found  to  be  identical  with  Entamoeba  tetragena. 
This  organism  was  found  only  in  cases  of  dysentery,  and  it  produced 
typical  ulcerating  dysentery  in  cats.  As  a  rule,  it  is  not  as  pathogenic 
for  cats  as  the  Entamoeba  histolytica.  In  all  cases  from  Africa  and 
South  America  which  Hartmann  examined  he  found  Entamoeba 
tetragena, 


INFECTIOUS  ENTEROHEPATITIS  IN  TURKEYS  549 

Werner  confirmed  Viereck's  and  Hartmann's  findings.  In  one  case 
he  observed  an  ameba  which  differed  from  Entamoeba  tetragena  and 
also  differed  somewhat  from,  but  closely  resembled,  Entamoeba 
hystolytica.  He  attempted  to  cultivate  the  pathogenic  ameba,  but 
failed.  He  was  able  to  get  amebse  to  grow,  but  always  found  that 
they  were  the  non-pathogenic  forms,  and  considers  them  to  have  been 
present  with  the  pathogenic  amebse  in  the  material  which  he  used 
in  making  his  cultures.  He  is  of  the  opinion  that  pathogenic  ameba? 
have  so  far  not  been  cultivated,  and  that,  therefore,  all  studies  of 
amebse  from  culture  have  been  of  non-pathogenic  amebae. 

Noc  studied  the  amebse  from  the  drinking  water  in  Saigon,  also 
the  amebse  from  the  stools  of  cases  of  dysentery  and  from  the  pus 
of  liver  abscesses  originating  in  Saigon.  He  was  able  to  cultivate 
the  amebse  from  these  sources,  and  found  that  he  had  the  same 
organism  in  the  drinking  water,  the  stools  of  cases  of  dysentery,  the 
dysenteric  ulcers  of  the  intestines,  and  the  pus  of  liver  abscesses. 
This  ameba  closely  resembled  Entamceba  hystolytica,  but  differed 
from  it  in  being  rich  in  nuclear  chroma  tin  and  in  forming  large 
polymorphous  cysts.  It  also  differed  from  Entamoeba  tetragena  and 
Entamoeba  coli. 

Infectious  Enterohepatitis  in  Turkeys. — Enterohepatitis  in  turkeys 
is  an  infectious  disease  apparently  due  to  a  pathogenic  protozoon 
called  Amoeba  meleagridis  by  Theobald  Smith,  its  discoverer.  Gush- 
man,  of  Rhode  Island,  in  1893,  noticed  a  peculiar  disease  among 
turkeys  which  has  since  been  found  in  various  other,  particularly 
Eastern,  States.  The  affection  is  characterized  by  diarrhea  and 
generally  progressive  emaciation,  and  a  dark  discolorization  of  the 
comb,  wattles,  and  the  skin  of  the  head,  from  which  the  common  name 
black-head  of  turkeys  is  derived.  The  disease  frequently  attacks 
young  birds,  and  it  may  take  a  more  acute  or  a  -markedly  chronic 
course.  The  most  important  pathologic  changes  are  the  following: 
The  ceca  of  the  birds  show  a  thickening  of  the  wall  and  a  superficial 
and  even  deep  destruction  of  the  mucosa  and  submucosa.  The 
thickening  may  be  uniform,  or  it  may  be  present  in  circumscribed 
places  only.  The  changes  are  generally  most  marked  near  the  blind 
ends  of  the  intestinal  pouches;  sometimes  the  cecum  only  is  diseased 
while  the  other  part  is  not  changed.  In  the  early  stages  the  adenoid 
tissue  between  the  pouches  and  in  the  submucosa  becomes  much  in- 
creased. With  the  extension  of  the  disease  much  of  the  mucous  mem- 
brane may  become  destroyed  by  ulcerative  and  desquamative  processes, 
and  fibrinous  material  is  deposited  on  the  denuded  intestinal  surface 
of  the  affected  intestines.  In  the  majority  of  cases  secondary  lesions 
are  found  in  the  liver,  which,  according  to  the  description  of  Smith  and 
Moore,  is  enlarged  to  perhaps  twice  its  normal  size.  On  the  surface 
are  seen  roundish  discolored  spots,  sharply  differentiated  from  the  rest 
of  the  tissue.  They  vary  from  3  to  15  mm.  in  diameter,  and  may  be 
lemon-yellow,  neutral-gray,  ochre-yellow,  or  of  a  mottled  brownish 


550  CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 

color.  They  are  not  elevated  but  rather  depressed,  and  contain 
necrotic  material  in  their  interior.  Such  foci  are  also  found  in  the 
depth  of  the  liver  tissue,  not  reaching  up  to  the  surface.  In  these 
areas,  as  in  the  cecal  pouches,  large  numbers  of  amebae  are  found. 
According  to  Smith's  observations  these  are  very  numerous  in  the 
affected  tissues  in  recent  cases  or  when  the  disease  is  at  its  height. 
They  disappear,  however,  from  areas  where  degenerative  and  necrotic 
changes  are  much  advanced.  The  most  frequent  appearance  pre- 
sented is  that  of  round  homogeneous  bodies  with  a  sharply  defined 
single  contour.  Within  the  parasites  situated  a  short  distance  away 
from  the  centre  is  a  group  of  minute  granules  probably  representing 
nuclear  chromatin.  The  Amoeba  meleagridis  is  generally  between 
8  to  10  micra  in  diameter,  sometimes  between  12  to  14  micra.  The 
tissue  reaction  against  the  invading  protozoa  leads  to  the  formation  of 
giant  cells,  and  the  parasites  are  often  seen  in  their  protoplasm.  This, 
however,  is  a  process  of  phagocytosis  on  the  part  of  the  tissue  cells, 
because  the  Amoeba  meleagridis  is  evidently  not  an  intracellular 
parasite,  but  it  penetrates  between  the  cells,  which  it  destroys  by  the 
intercellular  invasion.  The  life  cycle  of  this  organism  has  not  yet 
been  studied. 

BALANTIDIUM  COLI. 

The  subphylum  infusoria  is  not  of  great  importance  as  far  as 
microorganisms  pathogenic  to  higher  animals  and  men  are  concerned. 
From  what  is  known  today,  there  is  one  infusorium  which  may  produce 
disease  in  man  and  which  is  frequently  a  parasite  in  the  intestines  of 
the  hog,  from  which  it  probably  occasionally  invades  the  large  intes- 
tines of  man.  This  infusorium  is  the  Balantidium  coli.  It  can 
generally  be  obtained  for  the  purpose  of  study  in  the  intestinal  con- 
tents of  the  hog.  The  organisms  belong  to  the  subphylum  infusoria, 
class  ciliata,  order  heterotrichida,  family  bursaridse.  Infusoria 
possess  cilia  as  a  motor  apparatus  and  a  macronucleus  and  micro- 
nucleus.  The  members  of  the  family  to  which  balantidium  belongs 
generally  have  a  short,  pocket-like  body.  Their  chief  characteristic 
is  a  peristome,  which  is  not  a  mere  furrow,  but  a  broad,  triangular, 
deeply  excavated  area,  which  ends  at  a  point  at  the  mouth. 

The  body  of  Balantidium  is  oval  or  egg-shaped.  It  possesses  a 
short  gutter  or  funnel-shaped  peristome — which  is  continued  into  a 
short  esophagus.  The  body  is  externally  lined  by  a  fine  skin  or 
pellicula,  under  which  an  alveolar  ectoplasm  is  found.  The  whole 
exterior  is  covered  by  fine  cilia.  The  entoplasm  is  cloudy  and  contains 
droplets  of  mucus  and  fat.  In  it  two  contractile  vacuoles  are  also 
situated.  The  anus  of  the  infusorium  is  indicated  by  a  prominence 
in  the  protoplasm.  It  is,  however,  always  fused  except  during  the 
act  of  expelling  waste  material.  The  macronucleus  is  kidney-  or 
bean-shaped,  and  contains  much  chromatic  material.  The  micro- 


BALANTIDIUM  COLI 


551 


nucleus  is  small,  round,  and  vesicular.  The  size  of  Balantidium  coli 
varies  between  60  to  100  micra,  its  width  between  50  to  70  micra. 
Reproduction  occurs  by  division,  budding,  and  conjugation.  In 
division  the  macronucleus  divides  amitotically,  the  micronucleus 
mitotically.  Leuckard  has  described  encystation  of  the  parasite. 
If  this  is  to  occur  the  cilia  are  gradually  lost,  except  a  few  near  the 
mouth,  and  the  body  finally  becomes  perfectly  round  and  surrounds 
itself  with  a  heavy  capsule.  Encystation  has  also  been  noticed  after 
conjugation.  Very  probably  the  infection  is  transmitted  from  one 
host  to  another  in  the  encysted  stage  of  the  parasite.  The  organism, 
according  to  a  monograph  of  Strong,  has  been  found  in  about  150 
cases  in  man,  generally  in  cases  of  obstinate  diarrhea.  In  some  of 

FIG.  187 


Balantidium  coli:  1,  2,  stages  of  division;  3,  conjugation.     (After  Leuckart.) 

the  observed  cases  death  followed.  Most  authors  look  upon  balan- 
tidium  as  a  harmless  commensale  of  the  hog  and  occasionally  man, 
but  some,  like  Strong,  believe  that  it  may  be  the  cause  of  diarrhea! 
intestinal  disturbance  and  the  ulcerations  accompanying  it.  Strong 
and  others  have  found  the  balantidium  in  the  intestinal  tissues,  and 
there  is  no  doubt  that  the  organism  engulfs  and  digests  the  blood 
corpuscles  of  its  host.  The  author  has  seen  a  case  of  intense  balan- 
tidium infection  in  a  Filippino,  but  since  the  infusorium  was  associated 
with  an  uncinaria  infection,  it  was  impossible  to  decide  to  which 
of  the  two  the  pathologic  disturbance  was  mostly  due.  Brooks  has 
described  an  epidemic  among  the  orang-utangs  of  the  New  York 
Zoological  Garden,  in  which  balantidium  was  found  in  large  numbers 
in  the  stools.  Several  of  the  animals  died  and  the  postmortem 
examinations  showed  ulcerations  in  the  intestines  and  balantidium 
in  the  tissues. 


552          CLASSIFICATION  AND  MORPHOLOGY  OF  AMEBA 


QUESTIONS.  , 

1.  Give  the  proper  classification  of  ameba. 

2.  What  does  the  term  ameba  signify? 

3.  What  is  a  pseudopodium?    What  a  lobose  pseudopodium  ? 

4.  Describe  the  general  morphology  of  amebse. 

5.  What  methods  of  reproduction  have  been  observed? 

6.  What  is  meant  by  endoplasm,  ectoplasm,  chromidia  formation? 

7.  How  can  amebss  be  studied  in  the  live  state  ? 

8.  Describe  the  two  staining  methods  recommended  for  the  study  of  amebae. 

9.  Describe  the  culture  medium  used  in  cultivating  amebse. 

10.  What  is  a  pure-mixed  culture  of  amebse? 

11.  What  is  the  procedure  for  obtaining  a  growth  of  amebse:   (a)  in  the  case 
of  saprophytic  amebse,  (6)  in  the  case  of  parasitic  amebse  ? 

12.  Describe  in  detail  the  subsequent  steps  to  get  a  pure-mixed  culture  of 
amebae. 

13.  Do  amebse  enter  into  symbiotic  community  with  all  bacteria? 

14.  What  is  the  "hanging  plate  culture"  method  of  Walker? 

15.  Are  all  parasitic  amebae  pathogenic?    Name  some  pathogenic  amebse. 

16.  Describe  the  morphology  of  Entamreba  coli. 

17.  Describe  the  morphology  of  Entamceba  hystolytica. 

18.  Give  the  main  differential  morphologic  and  biologic  features  of  Entamoeba 
coli  and  Entamoeba  hystolytica. 

19.  Is  Entamoeba  coli  ever  found  in  the  intestines  of  healthy  man  ?    If  so, 
what  is  the  procedure  for  finding  it  ? 

20.  Name    some   other  amebae  (besides   Entamceba  hystolytica)  pathogenic 
to  man. 

21.  What  is  enterohepatitis  in  turkeys?    What  is  its  common  name? 

22.  In  what  organs  and  structures  are  the   main  pathologic  lesions  of  this 
disease  found? 

23.  Describe  the  chief  pathologic  lesions. 

24.  Name  and  describe  the   organism   causing   infectious  enterohepatitis  in 
turkeys. 

25.  What  kind  of  an  organism  is  Balantidium  coli? 

26.  Where  generally  found  ?     Does  it  ever  infect  man  ? 

27.  Is  it  ever  pathogenic  to  man  or  animals?    What  reasons  are  there  to  believe 
that  it  is  pathogenic  ? 


CHAPTEE    LIT. 

TRYPANOSOMES  AND  TRYPANOSOMIASES— CERCOMONAS— 
TRICHOMONAS— HERPETOMONAS. 

TRYPANOSOMES. 

Historical. — Trypanosomes  were  first  seen  by  Valentine  in  1841 
in  the  blood  of  trout,  and  in  the  following  two  years  they  were  found 
by  several  observers  in  the  blood  of  a  number  of  species  of  frogs. 
The  first  trypanosomes  in  a  mammal  was  discovered  by  Lewis  in 
India,  in  1878,  in  the  blood  of  the  rat.  Two  years  later  Evans,  chief 
veterinarian  of  the  English  Army  in  India,  discovered  trypanosomes 
in  the  blood  of  horses,  camels,  and  other  animals  sick  with  the  affection 
known  as  surra,  and  he  expressed  the  opinion  that  these  blood  para- 
sites were  the  cause  of  the  disease.  This  claim  was,  however,  at 
that  time  not  widely  accepted  and  the  study  of  trypanosomes  as  an 
etiologic  factor  of  disease  was  not  extensively  undertaken  until  Bruce, 
in  1894,  had  discovered  trypanosomes  as  the  cause  of  nagana  of 
horses  and  cattle  in  Africa.  Since  that  time  these  flagellata  have 
been  discovered  in  a  number  of  animal  diseases,  and  today  their  great 
importance  in  veterinary  and  human  pathology  is  well  established. 

Classification  and  Morphology. — Trypanosomes  belong  to  the  proto- 
zoan subphylum  mastigophora  (whip  carriers),  to  the  first  class 
(zoomastigophora)  in  which  animal  characteristics  are  predominant, 
and  they  form  the  fourth  order  of  this  class.  They  are  defined  by 
Calkins  as  follows:  "Organisms  of  elongated,  usually  pointed  form, 
and  a  parasitic  mode  of  life,  with  one  or  two  flagella  arising  from 
a  special  motor  nucleus,  and  with  an  undulating  membrane  provided 
with  myonemes  running  from  the  kinetonucleus  to  the  extremity  of 
the  cell;  one  of  the  flagella  is  attached  to  the  edge  of  this  membrane 
throughout  its  length,  and  may  terminate  with  the  membrane  or 
be  continued  beyond  the  body  as  a  free  lash." 

Trypanosomes  generally  have  an  elongated  spindle-,  lancet-,  or 
eel-shaped  protoplasmic  body;  sometimes  the  spindle  is  almost  as 
wide  as  it  is  long.  This,  however,  is  only  exceptionally  the  case,  and 
the  student  first  familiarizing  himself  with  trypanosomes,  particularly 
those  in  higher  vertebrates,  will  do  well  to  remember  them  as  little, 
rather  slender,  eel-shaped  bodies  of  the  size  of  an  involuntary  muscle 
cell  of  the  non-pregnant  mammalian  uterus.  Their  protoplasm  shows 
two  chromatic  or  nuclear  masses.  One  of  them,  as  a  rule,  placed  at  or 
near  the  centre  is  a  comparatively  large,  finely  granular  body,  called 


554 


TRYPANOSOMES  AND  TRYPANOSOMIASES 


the  nucleus  proper,  the  tropho-  or  the  macronucleus;  the  other  one, 
quite  small  and  dot-like,  is  known  as  the  micronucleus,1  or,  more 
properly,  the  centrosome,  or  blepharoblast,  and  it  is  generally  found 
at  the  posterior  end  of  the  microorganism.  From  the  centrosome 
starts  a  thin,  folded  membrane,  the  undulating  membrane,  it  has  a 
thickened  border  which  runs  out  into  a  free  whip-like  filament  called 
the  flagellum.  The  latter  is  composed  of  three  parts,  the  root  which 

FIG.  188 


Anterior  extremity 


Large  protoplasmic 
granules 


Nucleus 


Centrosome 
Posterior  extremity 


Flagellum,  third 
part;  free  end 


Flagellum  .second 
part 


Undulat.  membrane 


i       Flagellum,  first  part 


Morphology  of  Trypanosoma  Brucei  (schematic). 

arises  from  the  blepharoblast  and  extends  in  the  protoplasmic  body  as 
far  as  the  undulating  membrane,  the  second  portion  which  runs  along 
the  free  border  of  the  latter,  and  the  third  portion  which  is  the 
filamentous  free  end.  The  undulating  membrane  and  the  flagellum 
form  the  organs  of  locomotion  of  the  trypanosome,  and  they  enable 

1  Calkins  says:  "The  terms  macronucleus  and  micronucleus  are  frequently  used  to  designate 
the  trophonuclei  and  kinetonuclei  of  these  flagellates  (trypanosomes),  but  this  use  of  the 
term  micronucleus  is  greatly  to  be  deplored,  since  the  kinetonucleus  has  absolutely  no  analogy 
with  the  micronucleus  of  infusoria,  and  the  binucleate  condition  of  the  trypanosomes  is  to  be 
explained  upon  other  grounds  than  that  of  the  ciliates." 


METHOD  OF  EXAMINATION  IN  TRYPANOSOMA  INFECTION  555 

it  to  move  about  freely  and  actively  in  the  body  fluids  where  is  has 
its  usual  habitat. 

While  the  protoplasm  of  the  trypanosome  shows  a  certain  amount 
of  contractility,  its  motion  is  almost  exclusively  due  to  the  undulating 
membrane  and  the  flagellum,  and  it  is  in  the  direction  to  which  the 
free  whip-like  filament  points  that  the  organism  moves.  For  this 
reason  the  end  from  which  the  free  flagellum  projects  is  generally 
called  the  anterior  extremity.  A  few  species  of  trypanosomes  have 
a  free  flagellum  at  each  end,  but  this  is  an  exceptional  occurrence 
among  these  flagellata.  In  addition  to  the  structures  described  the 
protoplasm  of  trypanosomes  shows  larger,  smaller,  and  very  minute 
granules,  which  exhibit  special  staining  properties,  and  which  are 
often  arranged  in  rows  or  striae;  occasionally  clear  open  vacuoles  are 
seen  in  their  posterior  part. 

From  the  foregoing  description  it  may  be  inferred  that  these 
animal  microorganisms,  though  unicellular  -and  parasitic,  show  a 
high  degree  of  differentiation  into  a  number  of  morphologic  com- 
ponents. When  trypanosomes  multiply  they  do  so  by  a  longitudinal 
splitting.  The  micro-  and  macronucleus  divide  first,  and  the  process 
of  splitting  then  generally  extends  to  the  protoplasm,  the  undulating 
membrane,  and  the  flagellum.  The  macronucleus,  however,  never,  like 
the  nuclei  of  most  of  the  cells  of  higher  plants  and  animals,  shows 
the  formation  of  karyokinetic  figures,  but  it  divides  amitotically, 
that  is,  by  direct  division. 

Habitat. — The  usual  habitat  of  the  trypanosomes  is  the  blood  of 
vertebrate  animals.  In  certain  diseases  they  are  also  found  in  the 
lymph  and  in  the  cerebrospinal  fluid.  They  are,  as  far  as  is  known, 
strict  parasites  which  can  only  exist  and  multiply  within  another 
living  host.  There  is  no  evidence  that  trypanosomes  are  ever  found 
free  in  the  outside  world.  Unlike  the  hemosporidia  of  malaria  and 
the  piroplasma  of  Texas  fever,  trypanosomes  do  not  invade  and  live 
inside  the  red  blood  corpuscles,  nor  do  they  engulf  into  their  own 
protoplasm  and  digest  the  erythrocytes  of  the  blood  of  their  host, 
as,  for  instance,  the  amebse  of  dysentery  do.  They  simply  float 
about  in  the  blood  plasma  and  derive  food  for  existence  and  multi- 
plication by  processes  of  osmosis.  WTiile  trypanosomes  have  been 
found  in  the  blood  of  many  types  of  vertebrate  animals,  such  as 
batrachians,  reptiles,  fishes,  birds,  and  mammals,  nothing  would 
be  more  erroneous  than  to  consider  all  of  them  dangerous  disease- 
producing  parasites.  Quite  to  the  contrary,  most  trypanosomes  so 
far  discovered  are  harmless  blood  parasites  and  apparently  no  more 
dangerous  to  their  host  than  many  of  the  bacteria  which  inhabit  the 
integument,  the  gastro-intestinal  and  the  genito-urinary  tract  of  the 
higher  animals  and  man  as  commensales. 

Method  of  Examination  in  Trypanosoma  Infection. — Demonstration 
of  trypanosomes  in  infected  animals  is,  as  a  rule,  a  very  easy  matter, 
although  they  are  sometimes  so  scanty  that  it  may  be  necessary  to 


556  TRYPANOSOMES  AND  TRYPANOSOMIASES 

concentrate  them  by  preliminary  centrifuging.  Frequently,  however, 
it  is  only  necessary  to  obtain  a  drop  of  blood,  for  instance,  from 
the  ear  of  a  larger  animal  or  from  the  tip  of  the  tail  of  a  rat,  or  mouse, 
allow  it  to  fall  on  a  clean  slide  and  cover  it  with  a  cover-glass.  This 
simple  preparation  should  be  examined  at  once  with  the  microscope. 
The  first  search  can  be  made  with  a  low-power  lens,  16  mm.  or  two- 
thirds  inch  focus.  With  this  magnification  a  peculiar  agitation  among 
the  red  blood  corpuscles  can  frequently  be  seen  in  a  part  of  the  field. 
If  this  spot  is  placed  in  the  centre  the  very  characteristic  micro- 
organisms can  generally  be  seen  either  with  a  high-power  dry  or 
with  an  oil-immersion  lens.  In  fresh  preparations  trypanosomes  easily 
betray  their  presence  even  to  the  tyro  by  their  shooting,  darting,  or 
spiral  motions  in  the  blood,  and,  in  some  trypanosome  infections,  as,  for 
instance,  in  dourine  in  horses,  in  the  juice  expressed  from  the  patchy 
infiltrations  of  the  skin  or  the  inguinal  glands,  and  in  sleeping  sickness  in 
man  in  the  centrifuged  cerebrospinal  fluid.  If  the  morphology  of  the 
pathogenic  trypanosomes  is  to  be  studied  in  detail,  dry  stained  prepar- 
ations are  necessary.  The  most  useful  stains  are  generally  a  com- 
bination of  eosin  and  methylene  blue  as  found  in  the  stains  of  Roman- 
owski,  Leishman,  and  Wright.  The  last  named,  in  particular,  is 
very  satisfactory,  because  its  use  is  very  simple.  It  is  only  necessary 
to  make  a  blood  smear  on  a  cover-glass  or  slide;  the  smear  is  allowed 
to  become  air  dry,  and  Wright's  stain  is  poured  on  at  once.  The 
stain,  dissolved  in  methyl  alcohol  also  fixes  the  preparation.  The 
undiluted  stain  is  left  on  for  one  minute,  then  enough  distilled  water 
is  added  to  the  fluid  to  cause  it  to  show  a  dark  precipitate,  while  at 
the  same  time  the  pink  of  the  eosin  shows.  This  dilute  stain  is  left 
on  for  two  minutes,  then  the  preparation  is  washed  in  distilled  water 
for  thirty  to  sixty  seconds,  finally  it  is  dried  with  filter  paper  and 
examined  with  an  oil-immersion  lens. 

Artificial  Culture  Media. — The  artificial  cultivation  of  trypanosomes 
was  first  successfully  carried  out  by  Novy  and  McNeal,  whose  culture 
medium  consists  of  ordinary  agar  distributed  to  tubes  and  kept 
melted  at  50°  C.  Twice  the  volume  of  aseptically  collected  defi- 
brinated  rabbit's  blood  is  then  added  to  each  tube.  When  the  agar- 
defibrinated-blood  mixture  has  solidified  in  a  slanting  position  the 
condensed  water  is  inoculated  with  the  blood  of  an  infected  animal 
or  from  a  previous  culture.  This  comparatively  simple  culture 
medium  has  enabled  Novy  and  his  assistants  to  obtain  much  infor- 
mation about  the  morphology  and  multiplication  of  the  trypanosomes. 

The  rat  trypanosome,  Trypanosoma  Lewisii,  is  very  easy  to  cultivate; 
the  pathogenic  trypanosomes  are  more  difficult.  Novy,  McNeal,  and 
Hare,  however,  also  succeeded  in  obtaining  Trypanosoma  Brucei  and 
Trypanosoma  Evansii  in  artificial  culture,  while  Laveran  and  Mesnil 
cultivated  Trypanosoma  Brucei,  Trypanosoma  dimorphon,  and 
Trypanosoma  gambiense. 

Novy  and  Knapp,  while  working  with  the  flagellates  found  in  the 


PATHOLOGIC  CHANGES 


557 


gut  of  mosquitoes,  could  not  as  easily  obtain  trypanosomes  in  pure 
cultures  as  when  cultivating  them  directly  from  the  blood  in  which 
they  are  generally  the  only  live  microorganisms.  They  devised  the 
following  method,  which  gave  satisfactory  results:  By  means  of  a 
glass  spatula,  made  by  drawing  out  the  end  of  a  glass  rod,  a  little  of 
the  mixed  culture  of  flagellates  and  bacteria  derived  from  the  gut  is 
spread  in  a  series  of  streaks  over  six  Petri  dishes.  Ordinary  agar 
may  be  used  in  the  first  three  dishes,  since  the  desired  dilution  is  not 
attained  until  the  last  three.  The  Petri  dishesjused  must  be  so 
constructed  that  they  can  be  sealed  tightly  with  a  wide  rubber  band. 
The  sealed  dishes  are  set  aside  at  room  temperature  for  ten  to  twelve 
days.  The  last  two  plates  frequently  contain  isolated  colonies  of 
flagellates,  which  can  be  transplanted  in  the  usual  way  to  the  blood 
agar  test-tubes.  The  flagellates  occurring  in  mosquitoes  under  ordi- 
nary circumstances  are,  however,  not  true  trypanosoma  but  the 
nearly  related  genera  of  crithidia  and  herpetomonas. 


FIG.  190 


Trypanosoma  Lewisii,  non-pathogenic  rat 
trypanosome.  X  1000.  (Author's  prepara- 
tion.) 


Trypanosoma  Evansii,  the    cause  of   surra. 
X  1000.     (Author's  preparation.) 


Pathologic  Changes. — It  is  very  probable  that  pathogenic  trypano- 
somes cause  disease  by  produing  or  setting  free  in  the  blood  plasma 
certain  toxins,  but  practically  nothing  is  known  about  the  latter, 
and  they  have  not,  like  certain  bacterial  toxins  and  endotoxins, 
been  isolated.  The  most  characteristic  pathologic  changes  of  try- 
panosomiasis  are  generally  a  progressive  anemia,  with  disturbances 
of  circulation,  congestion,  infiltration,  and  edema,  and  periodical 
elevation  of  temperature,  combined,  in  the  later  stages,  with  pareses 
and  paraplegias.  The  animals  finally  suffer  from  rapidly  progressing 
emaciation,  which  terminates  fatally.  Apart  from  the  anemic  changes 
of  the  blood  and  the  bone  marrow,  and  from  the  presence  of  the 
trypanosomes  in  the  blood  and  other  body  juices,  there  are  no  morbid 


558 


TRYPANOSOMES  AND  TRYPANOSOMIASES 


FIG.  191 


anatomic  changes  specifically  characteristic  for  trypanosomiases ; 
enlargement  of  the  spleen  and  the  lymphatics,  however,  is  frequently 
present.  When  an  infected  animal  is  nearing  a  fatal  termination 
the  trypanosomes  seen  in  the  blood  are  usually  less  mobile  and  more 
granular  than  they  are  under  other  conditions. 

Diseases  Due  to  Pathogenic  Trypanosomes. — The  most  important 

diseases  of  animals  and  man  due  to  trypanosomes  are  the  following: 

Surra. — This   disease   is   due   to   the  Trypanosoma  Evansii,  and 

attacks   horses   and   cattle,  water  buffaloes  and  carabaos  in  India, 

China,  the  Philippine  Islands,  and  other  Asiatic  countries. 

Nagana. — This  disease  is  due  to  Trypanosoma  Brucei,  and  is 
prevalent  in  Africa,  among  horses,  cattle,  camels,  wild  buffaloes, 

antelope,  wildepests,  and  prob- 
ably also  elephants.  It  is  in- 
variably fatal  in  the  equidae 
and  dog,  but  may  terminate  in 
recovery  and  immunity  in  cattle. 
Allied  to  nagana  are  a  number 
of  other  trypanosomiases  in 
various  parts  of  Africa  described 
by  Koch  (German  East  Africa), 
Theiler  (Transvaal),  Brumpt 
(Ogaden),  Schilling,  Ziemann 
and  Martini  (Togo),  Button  and 
Todd  (Gambia),  and  Broden 
(Congo).  Some  of  these  infec- 
tions are  probably  identical  with 
nagana,  while  others  are  due 
not  to  the  Trypanosoma  Brucei, 
but  to  different  distinct  species. 
Of  the  latter  the  best  known  is 

a  disease  of  bovidae  of  South  Africa  described  by  Theiler  under  the 
name  of  gall  sickness,  or  galziekte.  It  is  due  to  a  trypanosome  first 
fully  described  by  Laveran,  and  named  in  honor  of  its  discoverer 
Trypanosoma  Theileri. 

Coder  as,  or  Mai  de  Caveras. — This  is  the  trypanosomiasis  (Try- 
panosoma equinum)  of  horses  in  South  Africa,  first  discovered  by 
Voges.  . 

Dourine. — A  somewhat  peculiar  position  among  the  trypanoso- 
miases is  held  by  dourine,  or  mal  du  coit  (Trypanosoma  equiperdum), 
of  the  equidse,  a  disease  transmitted  directly  from  individual  to  indi- 
vidual by  sexual  intercourse.  It  is  the  only  pathogenic  trypanosome 
infection  of  domesticated  animals  occurring  in  European  countries, 
such  as  Spain,  France,  Germany,  Switzerland,  Austria,  Hungary, 
Turkey,  and  the  Balkan  States.  The  trypanosome  of  dourine  was  first 
seen  in  1894  by  Rouget,  and  the  presence  of  these  parasites  in  this 
affection  was  later  established  beyond  doubt  by  the  researches  of 


Trypanosoma  gambiense  in  human  blood, 
cause  of  sleeping  sickness  in  man.  X  1000. 
(Author's  preparation.) 


DISEASES  DUE  TO  PATHOGENIC  TRYPANOSOMES 


559 


Schneider  and  Bufford,  Nocard  and  others.  In  1901  the  disease  was 
first  described  by  Salmon  as  having  been  found  in  the  United  States, 
and  in  1907  Rutherford,  Higgins,  and  Watson  gave  an  elaborate  and 


FIG.  192 


Magnified  head  of  tsetse  fly,  Glossina  morsitans.     (Author's  preparation.) 


lucid  account  of  their  observations  of,  and  experiments  with,  dourine 
infections  encountered  in  Northwestern  Canada. 

African  Sleeping  Sickness. — This  is  a  very  fatal  disease  of  human 
beings,  and  is  due  to  Trypanosoma  gambiense. 


560 


TRYPANOSOMES  AND  TRYPANOSOMIASES 


Method  of  Propagation. — Dourine  of  the  horse  tribe  probably  never 
spreads  except  through  coitus,  and  in  this  respect  it  differs  materially 
from  the  other  known  trypanosomiases,  which  are  propagated  through 
biting  insects,  particularly  flies.  Nagana  is  the  trypanosomiasis  in 
which  the  mode  of  transmission  by  blood-sucking  flies  has  been  most 
carefully  studied  by  Bruce  and  others.  It  is  the  much  discussed 
tsetse  fly  (Glossina  morsitans),  which  in  biting  and  blood-sucking 
spreads  nagana  from  infected  to  non-infected  animals.  The  tsetse 
fly  when  at  rest  can  easily  be  distinguished  from  other  biting  but 
harmless  flies.  Its  wings  almost  completely  overlap  like  the  blades 
of  a  pair  of  shears.  In  other  blood-sucking  flies  resembling  the  tsetse, 

FIG.  193 


Glossina  palpalis,  Rob.      X  3%. 

the  wings  when  the  insects  are  at  rest  are  always  more  or  less  separated. 
In  Africa  the  tsetse  fly  is  usually  found  in  low-lying,  hot,  humid 
regions ;  it  is  never  seen  far  from  water.  Even  in  the  so-called  African 
fly-belt  it  is  not  universally  found,  but  is  often  strictly  localized.  It 
bites  most  furiously  during  the  day,  less  during  the  evening,  rarely 
during  the  night.  Both  sexes  are  blood-sucking.  They  follow  the 
big  game  in  Central  and  South  Africa.  It  has  been  found  that  many 
wild  animals  in  Africa  harbor  Trypanosoma  Brucei  in  their  blood 
in  small  numbers.  These  animals  are  not  sick,  and  are  evidently  in 
a  certain  sense  immune  against  the  nagana  trypanosomes,  but  the 
latter  when  spread  by  the  tsetse  fly  to  domestic  animals  produce  the 
disease  in  its  virulent  form.  It  has  already  been  said  that  cattle  may 


METHOD  OF  PROPAGATION  561 

recover  from  nagana.  Whether  such  animals  which  have  recovered 
ever  become  entirely  free  from  trypanosomes  or  whether  they  retain 
them  in  small  numbers  without  detriment  to  themselves  is  a  different 
question.  The  author  during  his  stay  in  the  Philippine  Islands 
repeatedly  examined  microscopically  the  blood  of  a  Government 
herd  of  about  forty  carabaos.  These  animals  had  gone  through  an 
attack  of  surra,  had  apparently  recovered  from  it,  were  in  good 
flesh,  and  strong  and  able  to  work.  Examinations  were  frequently 
negative.  From  time  to  time,  however,  a  few  trypanosomes  were 
found  in  the  blood,  and  undoubtedly  non-immune  animals  may  be 
infected  from  apparent  immunes  harboring  such  trypanosomes  in 
their  blood,  without  detriment  to  themselves.  This  is  an  important 
matter  from  the  standpoint  of  prophylaxis. 

FIG.  194 


A  tsetse  fly  (Glossina  longipennis,  Corti,  from  Somaliland)   in  resting  attitude,  showing 
position  of  wings.      X  3^. 

• 

Tsetse  flies  infected  with  trypanosomes  are  dangerous  for  a  short 
time  only.  It  has  been  shown  by  Bruce  and  others  that  these  insects 
after  having  fed  on  an  infected  animal  can  only  spread  the  disease 
within  forty-eight  hours.  Carnivora  may  also  contract  nagana  by 
devouring  the  flesh  and  blood  of  infected  animals.  When  carnivora 
become  infected  in  this  manner  it  is  very  probable  that  this  is  brought 
about  through  injuries  in  the  buccal  mucous  membrane.  Musgrave 
and  Clegg,  who  experimented  with  horses,  dogs,  goats,  rabbits,  guinea- 
pigs,  monkeys,  and  cats,  have  shown  in  numerous  experiments  that 
feeding  trypanosoma-infected  blood  or  other  material  only  lead  to 
an  infection  in  the  presence  of  an  injury  to  the  mucosa  of  the  gastro- 
intestinal tract. 

The  question  presents  itself  whether  trypanosomes  in  the  body  of 
their  intermediary  host  (the  tsetse  fly)  undergo  a  cycle  of  life  changes 
36 


562  TRYPANOSOMES  AND  TRYPANOSOMIASES 

resembling  that  of  the  hemosporidia  of  malaria  in  the  body  of  the  ano- 
pheles mosquito.  Gray,  Tulloch,  and  Koch  have  claimed  that  ingested 
mammalian  trypanosomes  (Trypanosoma  gambiense  and  Brucei) 
undergo  such  developmental  changes  in  the  tsetse  fly,  but  Novy  has 
shown  that  what  these  investigators  believed  to  be  developmental 
male  and  female  sexual  forms  were  in  reality  a  different  species  of 
trypanosoma  parasitic  in  flies  and  mosquitoes  and  in  no  way  connected 
with  the  pathogenic  trypanosoma  of  nagana.  Neither  surra  nor 
nagana,  the  two  most  important  trypanosomiases  spread  by  biting 
flies,  have  even  been  found  in  man.  There  is,  however,  one  human 
infection,  the  celebrated  African  sleeping  sickness,  due  to  the  Try- 
panosoma gambiense,  and  mentioned  briefly  above,  which  is  spread 
by  biting  flies,  and  occurs  over  a  large  territory.  It  was  first  described 
over  a  hundred  years  ago  by  Winterbottom,  and  its  cause,  a  trypano- 
some,  was  discovered  by  Dutton  in  1901.  The  Trypanosoma  gam- 
biense of  human  sleeping  sickness  transmitted  by  Glossina  palpalis 
is  pathogenic  for  a  large  number  of  animals,  such  as  the  monkey, 
lemur,  dog,  jackal,  cat,  rabbit,  guinea-pig,  etc. 

Trypanosoma  Americanum,  n.  sp. — Crawley  has  recently  reported 
that  he  has  obtained  from  cultures  prepared  from  the  blood  of  normal 
cattle  a  new  hitherto  undescribed  species  of  trypanosoma,  which, 
however,  is  not  present  in  the  blood  of  the  animals  as  such,  but  in 
an  unknown  form,  which  only  develops  into  typical  trypanosomes  in 
the  artificial  cultures.  Crawley  has  named  this  non-pathogenic 
flagellate,  Trypanosoma  Americanum  (novum  species).  According 
to  the  observer  the  organism  can  be  demonstrated  as  follows :  Blood 
is  drawn  under  all  aseptic  precautions  from  the  jugular  vein  of  a 
cow  by  means  of  a  sterile  syringe  and  transferred  to  flasks  of  100  c.c. 
capacity.  About  30  to  50  c.c.  of  blood  is  taken  in  each  case.  In 
each  flask  are  placed  six  to  eight  common  faceted  beads  of  rough 
glass  such  as  are  used  for  cheap  necklaces.  The  flasks  containing 
the  blood  and  the  beads  are  then  shaken  for  a  few  minutes,  which 
causes  the  fibrin  to  collect  around  the  beads.  The  defibrinated 
blood  is  distributed  to  flasks  and  tubes  containing  nutrient  (beef) 
bouillon.  Muttori  bouillon  may  also  be  used.  The  inoculated  flasks 
and  tubes  are  kept  at  room  temperature.  After  two  or  three  days 
trypanosomes  appear,  and  after  a  few  more  days  they  can  be  seen 
without  the  aid  of  the  microscope  as  little  colonies  on  the  surface 
of  the  column  of  blood  cells.  Those  colonies  show  as  small,  white 
plates,  and  may  be  3  to  4  mm.  in  diameter.  They  are  readily  dis- 
tinguishable from  bacterial  contamination  in  that  they  are  flat  and 
sharply  circumscribed,  while  the  masses  of  bacteria  are  always 
more  or  less  diffuse  and  tend  to  cloud  the  bouillon.  These  trypano- 
somes are  not  present  as  such  in  the  freshly  drawrn  blood  of  cattle, 
but  the  evidence  is  that  they  develop  in  the  cultures  from  round 
or  oval  bodies.  From  these  lenticular  bodies  are  first  formed,  then 
a  flagellum  is  developed,  and  the  flagellate  now  formed  is  of  the 


TRYPANOSOMES  IN  BIRDS  563 

type  of  crithidia,  these  elongate,  become  trypanosome-like  and  later 
are  endowed  with  an  undulating  membrane.  After  the  typical 
trypanosome  shape  has  been  developed  a  trophonucleus  is  seen  as  a 
fair-sized  vesicle,  containing  coarse  chromatin  granules.  The  kineto- 
nucleus  (micronucleus)  is  usually  elongated,  forming  a  long  ellipsoid, 
and  stains  an  almost  black-garnet  color.  The  two  nuclei  are  always 
close  together.  The  undulating  membrane  is  well  developed.  The 
parasites  appear  under  two  forms,  a  band-shape  and  a  club-shape. 
The  band-shaped  forms  are  more  typical  trypanosomes,  and  are  more 
numerous  than  the  others.  In  all  the  cultures  the  trypanosomes  tend 
to  occur  in  great  clusters.  These  must  not  be  confounded  with 
agglutination  rosettes,  which  present  a  wholly  different  appearance. 
In  the  clusters  nothing  in  the  way  of  a  definite  orientation  can  be 
made  out,  while  in  the  agglutinations  seen  the  arrangement  was 
radial. 

Observations  similar  to  those  made  by  Crawley  had  previously 
been  made  on  Japanese  cattle  by  Miyajima,  who,  however,  believed 
that  the  trypanosome  which  he  obtained  in  cultures  represented  a 
stage  in  the  life  cycle  of  a  non-pathogenic  piroplasma.  The  obser- 
vations of  Miyajima  and  Crawley  as  to  the  development  of  trypano- 
somes from  healthy  cattle  when  their  blood  is  mixed  with  nutrient 
bouillon  need  further  confirmation  before  they  can  be  fully  accepted. 
It  is  not  to  be  forgotten  that  the  blood  platelets  when  blood  is  mixed 
with  certain  culture  media  often  assume  shapes  which  may  be 
easily  mistaken  for  trypanosomes.1 

Trypanosomes  in  Birds. — Trypanosomes  in  birds  have  been  studied 
extensively  by  Novy  and  MacNeal.  They  undertook  these  studies 
particularly  on  account  of  Schaudinn's  claim  that  trypanosomes  in 
birds  were  a  stage  in  the  life  history  of  the  avian  intracorpuscular 
parasite  halteridium  (Hsemoproteus  noctuse).  Schaudinn,  on  allowing 
the  common  mosquito  (Culex  pipiens)  to  feed  upon  the  blood  of  owls 
infected  with  halteridium,  found  in  the  intestines  of  about  10  per  cent, 
of  these  insects  large  numbers  of  trypanosomes  which  he  considered  a 
cycle  in  the  life  stage  of  the  Hsemocytozoon  halteridium.  Novy  and 
MacNeal  cultivated  such  trypanosomes  from  mosquitos  in  test-tubes 
on  the  blood-agar  medium.  The  trypanosomes  multiplied  readily,  but 
the  hematozoa  died  out.  When  birds  were  infected  with  such  pure 
cultures  of  trypanosomes  no  intracellular  parasites  developed.  The 
cultivation  of  the  bird  trypanosomes,  according  to  Novy  and  MacNeal, 
is  as  easy  as  that  of  the  rat  trypanosome.  They  concluded  from  their 
studies  that  trypanosome  infection  is  very  common  and  widespread 
in  birds,  that  different  avian  species  harbor  different  trypanosomes, 
and  that  one  bird  may  be  infected  by  several  species  of  trypanosomes. 
The  latter  are  not  pathogenic  for  birds,  but  evidently  harmless  para- 
sites. 

1  Swingle  has  recently  called  attention  to  this  fact  in  a  paper  published  in  the  Journal  of 
Infectious  Diseases. 


564  CERCOMONAS,  TRICHOMONAS,  HERPETOMONAS 


CERCOMONAS— TRICHOMONAS— HE  RPE  TOM  ON  AS. 

While  trypanosomes  are  the  most  important  flagellate  from  a 
medical  and  veterinary  standpoint,  there  are  a  few  others  which 
are  parasites  of  vertebrate  animals,  though  most  of  them  are  not 
pathogenic. 

Gercomonas. — These  are  flagellates  of  a  round  or  oval  body,  with 
a  pointed  posterior  end.  The  flagella  are  generally  long.  The 
vesicular  nucleus  is  situated  near  one  or  two  contractile  vacuoles. 
Cercomonas  hominis,  first  described  by  Davaine,  1854,  has  been 
found  in  the  human  intestines,  urine,  and  the  sputum  in  disease  (gan- 
grene) of  the  lung.  The  organism  is  pear-shaped,  with  a  pointed 
posterior  end,  from  10  to  12  micra  long;  the  flagellum  is  twice  as 
long  as  the  main  body.  The  nucleus  is  difficult  to  see.  The  organism 
is  most  frequently  found  in  the  intestines  of  man  in  chronic  diarrheas. 


FIG.  195 


FIG.  196 


Trichomonas  vaginalis.     (Blochmann.) 


Lamblia  intestinalis.     (Schewiakoff.) 


It  is,  however,  not  the  cause  of  the  pathologic  condition,  but  only 
finds  in  the  fluid  contents  of  the  large  intestines  conditions  favorable 
to  its  parasitic  existence.  The  following  organisms  of  this  family 
have  been  found  in  domestic  and  other  animals:  Cercomonas  anatis 
(duck),  Cercomonas  canis  (dog),  Cercomonas  gallinarum  (chicken). 
Several  species  of  cercomonas  have  been  found  in  the  intestines  of 
guinea-pigs;  two  of  them,  Cercomonas  pisiformis  and  Cercomonas 
globulus,  are  believed  to  be  pathogenic  for  this  animal. 

Trichomonas. — Trichomonas  vaginalis. — This  organism  is  found  in 
the  vaginal  secretion  and  in  the  urine  of  females,  occasionally  also  in 
the  urethra  and  urine  of  males.  The  organism  is  either  pear-shaped 
or  circular.  It  varies  much  in  size,  and  the  measurements  given  are 
from  10  to  25  micra;  generally  in  length  from  15  to  25  micra,  and  the 
width  from  7  to  12  micra.  The  protoplasm  is  finely  granular  and  of 
a  greenish  hue.  The  anterior  end  of  the  cell  carries  three,  sometimes 
four,  flagella,  which  are  about  10  micra  long. 

Trichomonas  Hominis,  or  Intestinalis. — This  is  generally  smaller 
than  the  preceding  one.  It  is  a  parasite  of  the  ga.stro-intestinal 


HERPETOMONAS  565 

tract  of  man.  It  is  particularly  found  in  chronic  diarrheas,  but  it 
is  not  pathogenic.  Prowazek  found  a  similar  trichomonas  in  monkeys 
and  other  animals. 

Lamblia  Intestinalis . — This  is  another  organism  of  the  order  poly- 
mastigina,  and  is  found  in  the  intestines  of  mice,  rats,  dogs,  cats, 
sheep,  and  rabbits,  and  occasionally  of  man.  The  organism  is  beet- 
shaped  and  bilaterally  symmetrical.  It  is  10  to  21  micra  long,  5  to 
12  micra  wide ;  the  flagella  measure  from  9  to  14  micra.  At  the  anterior 
end  the  organism  possesses  a  kind  of  sucking  concavity,  the  margins 
of  which  project  and  appear  to  be  contractile.  It  has  four  pairs  of 
flagella,  arranged  one  pair  as  anterior,  one  pair  as  posterior,  and 
two  pairs  as  lateral  flagella.  The  posterior  portion  of  the  body  forms 
a  tail  2  to  2J  micra  long,  from  which  the  posterior  flagella  project 
outward.  The  nucleus  is  bilaterally  symmetrical,  and  each  half  is 
oval  in  shape,  each  side  generally  contains  a  deeper  staining  nucle- 
olus-like  body.  The  protoplasm  is  densely  hyaline  and  surrounded 
by  a  kind  of  external  pellicle.  Contractile  vacuoles  are  not  present. 
The  exact  mode  of  division  is  unknown,  but  cysts  surrounded  by 
a  chitinous  membrane  and  possessing  four  nuclei  have  been  observed. 
Infection  of  men  and  animals  is  brought  about  by  ingestion  of  the 
cysts.  Grassi  has  demonstrated  this  mode  of  infection  by  swallowing 
cysts.  Lamblia  in  the  intestines  fastens  itself  by  the  sucking  apparatus 
at  its  anterior  end  to  the  intestinal  epithelia.  The  organism,  however, 
does  not  appear  to  be  pathogenic.  Parts  of  the  intestines  where 
numerous  lamblia  are  attached  do  not  show  any  pathologic  changes. 

L.  Pfeiffer  found  numerous  trichomonas-like  protozoa  in  the 
intestines  of  chickens,  ducks,  and  other  birds  suffering  from  diarrhea 
with  diphtheritic  inflammatory  changes  in  the  intestines.  It  has, 
however,  not  been  conclusively  proved  that  these  trichomonas,  or 
lamblia,  were  the  cause  of  the  intestinal  pathologic  changes  and  the 
death  of  the  fowl. 

Herpetomonas. — Herpetomonas,  which  are  nearly  related  to  the 
cercomonas  on  one  side  and  to  the  trypanosomes  on  the  other,  are 
described  by  Kent  as  free  swimming,  elongate  or  vermicular,  highly 
flexible;  the  posterior  extremity,  often  the  most  attenuate,  but  not 
constituting  a  distinct  caudal  appendage;  flagellum  single,  terminal. 
Herpetomonas  are  intestinal  parasites  of  flies  and  other  insects. 
The  best-known  species  is  the  Herpetomonas  muscse  domes tica, 
the  intestinal  parasite  of  the  common  house  fly.  It  has  been  studied 
by  Prowazek,  who,  according  to  Calkins,  describes  it  as  follows: 
"This  organism  is  elongate  and  somewhat  flattened  at  one  end, 
which  gives  rise  to  the  single,  long,  vibratile  flagellum.  Apart  from 
the  nucleus  and  blepharoblast  the  inner  protoplasm  has  no  char- 
acteristic structures,  and  the  nucleus  is  of  the  characteristic  masti- 
gophora  type,  with  chromatin  granules  of  more  or  less  definite  number. 
The  blepharoblast  lies  between  the  nucleus  and  the  flagellum,  and  is 
frequently  of  large  size,  while  from  it  the  base  of  the  flagellum  takes 


566 


CERCOMONAS,  TRICHOMONAS,  HERPETOMONAS 


its  origin."  At  the  base  of  the  flagellum,  just  outside  of  the  body, 
is  a  small  basal  granule.  Reproduction  occurs  by  longitudinal  division. 
The  nucleus  divides  by  a  primitive  process  of  mitosis,  the  granules 
being  equally  distributed.  This  nuclear  division  is  preceded  by 
division  of  the  blepharoblast  and  of  the  flagellum,  which  in  this 
case  appears  to  divide  throughout  its  entire  length,  instead  of  one 
being  formed  as  in  some  trypanosomes  by  outgrowth  from  the  blephar- 
oblast. Conjugation  has  been  described  by  Prowazek  as  taking  place 
between  forms  which  are  not  sexually  differentiated  beyond  the 
fact  that  one  appears  to  be  denser  and  larger  than  the  other.  After 
conjugation  a  permanent  resting  cyst  is  formed  by  the  fertilized 
cell,  and  in  this  condition  the  parasite  passes  from  the  intestine  with 
the  feces  of  the  host.  Infection  of  new  hosts  usually  takes  place 
by  ingestion  of  these  permanent  cysts  with  the  food. 


FIG.  197 


Herpetomonas  Donovani,  unequal  division  to  form  slender  flagellated  individuals. 
(After  Leishman.) 

The  genus  herpetomonas  has  assumed  considerable  importance, 
first,  in  consequence  of  Schaudinn's  claim  that  the  hemosporidia  of 
birds  are  a  stage  in  the  life  cycle  of  herpetomonas,1  and  secondly,  of 
greater  importance,  because  it  is  now  known  that  a  herpetomonas  is 
the  cause  of  a  widespread  tropical  disease  of  man. 

This  disease,  which  occurs  in  India,  China,  Egypt,  Arabia,  Tunis, 
Algiers,  and  other  tropical  countries,  is  known  as  kala  azar,  tropical 
febrile  splenomegaly,  dum-dum  fever,  and  by  other  names.  Leish- 
man discovered  peculiar  bodies  in  the  spleen  of  a  person  dead  from  the 
disease  and  his  observation  was  first  confirmed  by  Donovan  and  later 
by  others,  including  Marchand,  Rodgers,  and  Christopher.  These 
bodies,  first  known  as  the  Leishman-Donovan  bodies,  are  found  in 
great  numbers  in  the  large  cells  of  the  spleen  or  liver.  They  are 
round  or  oval,  or  rather  cockle-shell-shaped,  and  have  two  chro- 


1  This,  as  already  explained,  is  denied  by  Novy  and  his  associates. 


QUESTIONS  567 

matin  masses  which  are  called  the  macro-  and  the  micronucleus. 
Rodgers,  in  1904,  succeeded  in  cultivating  these  bodies  in  blood 
from  the  spleen  mixed  with  normal  salt  solution  and  neutralized  or 
made  faintly  acid  by  the  addition  of  sodium  citrate  solution.  In 
such  artificial  cultures  the  Leishman-Donovan  bodies  developed  into 
flagellates  of  the  type  of  herpetomonas,  and  they  have  been  called 
Herpetomonas  Donovani  by  Laveran  and  Mesnil.  It  was  also 
subsequently  ascertained  that  the  bed-bug  (Cimex  rotundatus), 
in  whose  intestines  the  Leishman-Donovan  bodies  develop  into  the 
flagellate  type,  transmit  the  disease  from  the  sick  to  the  healthy. 
Wright  found  similar  intracorpuscular  bodies  in  the  granulation  tissue 
of  a  tropical  ulcer.  But  these  have  only  been  studied  in  sections,  and 
have  not  yet  been  cultivated  or  observed  in  a  flagellate  stage. 

QUESTIONS. 

1.  What  is  the  position  of  trypanosomes  in  the  phylum  protozoa? 

2.  What  does  the  word  mastigophora  mean  ? 

3.  Give  a  general  definition  of  trypanosomes. 

4.  Describe  their  morphology. 

5.  Define  the  terms :  trophonucleus,  kinetonucleus,  undulating  membrane. 

6.  Describe  the  origin  and  the  three  parts  of  the  flagellum  of  typical  trypan- 
osomes. 

7.  Where  are  trypanosomes  found?    Are  they  all  pathogenic? 

8.  Are  they  intra-  or  extracorpuscular  blood  parasites? 

9.  Describe  in  detail  the  method  to  examine  trypanosomes  in  the  live  state. 

10.  Likewise  the  method  to  examine  stained  specimens. 

11.  In  what  trypanosomiases  are  the  parasites  not  found  in  the  blood  but 
elsewhere  ?    Where  are  they  found  in  these  diseases  ? 

12.  Describe  method  of  obtaining  trypanosomes  in  pure  culture. 

13.  Which  vertebrate  trypanosome  can  be  easiest  cultivated  ? 

14.  What  is  the  method  of  obtaining  pure  cultures  of  flagellates  from  the  guts 
of  mosquitoes? 

15.  Who  discovered  the  first  pathogenic  trypanosome;  in  what  disease  was   it 
found,  and  where? 

16.  What  are  the  most  important  pathologic  changes  due  to  infection  by 
pathogenic  trypanosomes  ? 

17.  What  organism  causes  surra?     What  animals  are  susceptible  to  surra? 

18.  What  disease  is  caused  by  Trypanosoma  Brucei;  where  found,   what 
animals  are  susceptible  ? 

19.  What  is  dourine  ?    What  causes  this  disease  ?    How  is  it  transmitted  ? 

20.  Discuss  the  general  method  of  transmission  of  trypanosomiases. 

21.  Discuss  immunity  in  trypanosomiases. 

22.  Describe  the  life  cycle  of  trypanosomes  in  the  body  of  the  tsetse  fly. 

23.  What  disease  is  caused  by  Trypanosoma  gambiense? 

24.  What  is  Trypanosoma  Americanum,  n.  sp.  ? 

25.  Describe  its  morphology  in  artificial  cultures  and  in  the  circulating  blood 
of  cattle. 

26.  Are  birds  frequently  infected  with  trypanosomes?    What  diseases  do  the 
latter  cause  in  domestic  and  other  birds  ? 

27.  What  is  the  claim  of  Schaudinn  as  to  the  life  cycle  of  bird  hemosporidia 
after  their  entrance  into  the  gut  of  the  mosquito  ?    Discuss  this  claim. 

28.  What  is  cercomonas?    Describe  its  morphology  and  its  parasitic  properties. 

29.  Describe  the  morphology  and  parasitic  properties  of  Trichomonas  vaginalis 
and  intestinalis. 

30.  Describe  the  morphology  and  parasitic  properties  of  Lamblia  intestinalis. 

31.  Discuss  the  position  in  classification  and  the  morphology  of  herpetomonas. 

32.  Where  are  herpetomonas  generally  found  as  parasites? 

33.  Are  any  of  the  herpetomonas  pathogenic,  if  so,  describe  this  pathogenic 
organism  and  name  the  disease  it  causes. 


CHAPTEE    LIIL 

PATHOGENIC  SPOROZOA— COCCIDIA—HEMOSPORIDIA— 
MICROSPORIDIA— SARCOSPORIDIA. 

CALKINS  defines  the  subphylum  sporozoa  as  parasitic  protozoa 
without  motile  organs,  but  capable  of  moving  from  place  to  place 
by  structural  modifications  of  one  kind  or  other;  reproduction,  either 
simple  or  multiple,  but  mainly  by  spore  formation,  which  is  either 
asexual  (schizogony)  or  sexual  (sporogony).  This  subphylom  is  very 
rich  in  genera  divided  into  a  number  of  classes  and  orders.  Only 
four  orders,  however,  come  within  the  scope  of  this  work,  namely: 

A.  Coccidia. — These   are   cell-infecting   protozoa,   which    usually 
reproduce  by  schizogony  and  sporogony,  thus  giving  a  life  cycle  with 
an  alternation  of  asexual  and  sexual  generations.    After  fertilization 
the  oosphore  forms  sporoblasts  (mother  cells  giving  rise  to  spores), 
which  may  or  may  not  (asporocystea)   be  covered   by  a  sporocyst 
membrane,  and  which  may  each  become  transformed  into  one  or 
several  young  reproductive  spores  (sporozoites). 

B.  Hemosporidia. — These   are   intracorpuscular   blood    parasites 
which  may  change  from  a  permanent  to  an  intermediate  host,  or 
which  may  be  confined  to  one  host. 

C.  Microsporidia. — The  young  vegetative  cells  are   more   or  less 
ameboid;  the  spores  are  very  minute,  pyriform,  with  only  one  capsule, 
which  is  invisible  in  the  fresh  state.    They  are  intracellular  parasites 
of  invertebrates. 

D.  Sarcosporidia. — These  are  sporozoa  in  which  the  initial  stage 
is  passed  in  muscle  cells  of  vertebrates. 


COCCIDIA. 

Coccidia  are  all  cell  parasites;  they  are  found  in  vertebrate  and 
also  in  lower  animals,  such  as  mollusks  and  insects.  They  are  para- 
sitic in  the  cells  of  the  gastro-intestinal  and  geni to-urinary  tract. 
They  are  generally  round  or  oval.  The  protoplasm  does  not  show 
a  differentiation  into  endoplasm  and  ectoplasm.  As  a  rule,  it  contains 
granules,  which  differ  in  staining  affinities  toward  various  stains. 
The  nucleus  is  usually  situated  in  the  centre,  is  vesicular,  and  contains 
in  its  interior  a  granule,  which  has  received  the  name  karyosome. 
Propagation  occurs  alternately  in  sexual  and  then  asexual  manner, 
so  that  a  definite,  complicated  cycle  of  life  is  formed.  The  spores 


COCCIDIA 


569 


(merozoites)  developed  after  asexual  schizogony  spread  the  infection 
within  the  same  host  (auto-infection),  while  the  sporozoites  formed 
after  sexual  reproduction  may  spread  the  infection  to  a  new  host. 

The  life  cycle  of  a  coccidium  may,  therefore,  be  outlined  as  follows 
(Doflein,  Schaudinn) :  A  cyst  containing  sporozoites,  the  product  of 
sexual  propagation,  is  taken  into  the  gastro-intestinal  tract  of  an  animal. 


FIG.  198 


Life  cycle  of  Coccidium  schubergi.  (After  Schaudinn.)  Sporozoites  penetrate  epithelial 
cells  and  grow  into  adult  intracellular  parasites  (a).  When  mature  the  nucleus  divides  re- 
peatedly (6),  and  each  of  its  subdivisions  becomes  the  nucleus  of  a  merozoite  (c).  These  enter 
new  epithelial  cells,  and  the  cycle  is  repeated  many  times.  After  five  or  six  days  of  incuba- 
tion the  merozoites  develop  into  sexually  differentiated  gametes;  some  are  large  and  well 
stored  with  yolk  material  (d,  e,  f ) ;  others  have  nuclei  which  fragment  into  many  smaller  par- 
ticles ("Chromidia"),  each  granule  becoming  the  nucleus  of  a  microgamete,  or  male  cell  (d), 
h,  i,  j).  The  macrogamete  is  fertilized  by  one  microgamete  (0),  and  the  copula  immediately 
secretes  a  fertilization  membrane,  which  hardens  into  a  cyst.  The  cleavage  nucleus  divides 
twice,  and  each  of  the  four  daughter  nuclei  forms  a  sporoblast  (k)  in  which  two  sporozoites  are 
produced  (I). 

The  cyst  wall  or  membrane  is  dissolved,  the  sporozoites  become  free, 
and  a  number  of  them  enter  epithelial  cells,  penetrate  into  their 
nuclei,  and  grow  into  forms,  which  in  some  coccidia  already  show  a 
differentiation  into  cells  that  will  later  furnish  the  male  and  others 
that  will  furnish  the  female  gametes.  Both  kinds  of  cells  then  divide 
asexually  and  form  male  and  female  gametes,  respectively.  These 


570 


PATHOGENIC  SPOROZOA 


may  invade  new  epithelial  cells  of  the  same  host,  and  may  again 
subdivide  in  an  asexual  manner.  After  this  has  occurred  a  number 
of  times  the  female  merozoites  begin  to  grow  and  to  accumulate 
nutritive  material  in  their  protoplasm.  They  then  fall  out  of  the  cell 
which  they  have  infected  and  destroyed,  and  they  reduce  the  amount 
of  their  nuclear  chromatin  by  the  formation  of  polar  bodies  and  the 
expulsion  of  some  chromatin.  These  are  the  phenomena  of  maturation 
through  which  a  germ  cell  must  always  pass  before  it  can  be  fertilized 
by  another  germ  cell,  which  likewise  has  had  to  go  through  the  same 
process  of  maturation.  While  the  female  merozoites  have  in  this 
manner  become  the  macrogametes,  the  male  merozoites  have  gone 
through  a  series  of  nuclear  divisions  and  have  formed  a  number  of 
spindle-shaped  flagellated  microgametes .  The  latter  penetrate  into  the 
macrogametes,  and  this,  of  course,  constitutes  the  act  of  fertilization. 
The  copula  formed  by  the  union  of  the  microgametes  and  macrogam- 
etes later  divides  into  two  sporoblasts,  or  mother  cells,  giving  rise 
to  the  sporozoites,  which  are  the  result  of  the  sexual  fertilization  and 
propagation  which  have  occurred.  The  oocyst  containing  the  spores 
may  again  be  taken  up  by  a  new  host,  and  the  cycle  can  begin  anew. 

FIG.   199 
a  b  c  d  e  f  a  h  i 


Showing  spore  formation  in  Coccidium  cuniculi,  from  the  liver  of  a  rabbit:  a  and  6,  young 
stage  in  the  epithelial  cells  of  the  gall-ducts  (the  small  oval  is  the  cell  nucleus);  c,  d,  and  e, 
the  fertilized  oocyst;  in  d  the  protoplasm  is  beginning  to  shrink  away  from  the  cyst  wall,  and 
in  e  it  has  contracted  into  a  spherical  form;  f,  segmentation  into  four  sporoblasts;  g,  elongation 
of  the  sporoblasts  to  form  spores;  h,  four  complete  spores  in  the  oocyst;  i,  single  spore  more 
highly  magnified,  showing  the  two  sporozoites  and  a  small  quantity  of  residual  protoplasm. 
The  life  cycle  has  been  fully  worked  out  by  Simon.  (After  Balbiani,  from  Doflein.) 

Coccidium  Cuniculi. — The  best  and  probably  earliest  known  coccid- 
ium  pathogenic  to  mammals  is  the  Coccidium  cuniculi,  or  oviforme, 
first  described  as  Psorospermium  cuniculi  by  Rivolta  (1878).  It 
is  parasitic  in  the  intestinal  epithelium  and  the  liver  cells  of  wild 
and  tame  rabbits,  and  it  sometimes  causes  fatal  epidemics  among 
rabbits  of  laboratories  and  places  where  they  are  bred.  The  spore 
cysts  are  taken  up  with  food  soiled  by  feces  from  animals  harboring 
the  infection.  After  the  cyst  membrane  has  become  dissolved  the 
sickle-shaped  spores  penetrate  into  the  interior  of  the  intestinal 
epithelial  cells  of  the  host.  The  asexual  forms  are  20  to  50  micra 
long  and  20  to  35  micra  wide;  30  to  200  merozoites  are  formed  in 
asexual  reproduction.  The  coccidia  disease  of  rabbits  lasts  from 
one  to  two  weeks,  and  leads  to  fever,  diarrhea,  and  emaciation,  with 


COCCIDIOSIS  IN  CATTLE  AND  OTHER  ANIMALS  571 

a  yellowish  mucopurulent  discharge  from  the  mouth  and  nose.  The 
liver  is  very  much  enlarged,  and  shows  on  section  densely  crowded, 
grayish-white  nodules  from  the  size  of  a  millet  seed  to  a  hazelnut, 
These  nodules  are  surrounded  by  a  capsule,  and  often  contain  a  smeary 
mass  composed  of  degenerated  liver  epithelia,  leukocytes,  and  the 
pathogenic  coccidia.  The  invasion  occurs  from  the  bile-ducts,  the 
epithelial  cells  of  which,  while  being  destroyed  in  some  places,  pro- 
liferate in  other  places  as  a  result  of  the  inflammatory  stimulus. 
Rabbits  which  recover  from  the  infection  contain  the  oocysts  for  a  long 
time  in  their  appendix  and  their  gall-bladder.  Cicatrix  formation  and 
calcification  in  the  liver  are  often  seen  after  coccidiosis  in  rabbits. 

Another  coccidium  parasitic  in  the  intestines  of  rabbits  has  been 
named  Coccidium  perforans  by  Leuckart. 

Coccidiosis  in  Cattle  and  Other  Animals. — There  is  a  disease  of  cattle 
known  as  coccidiosis  intestinalis,  dysenteria  coccidiosa  bovum,  "Rothe 
Ruhr  der  Rinder"  (German),  "flux  de  sang"  (French),  characterized 
by  bloody  discharges  from  the  bowels,  without  fever,  but  with  pro- 
gressive emaciation  in  severe  cases.  The  disease  occurs  particularly 
among  young  animals  and  on  marshy  pastures.  Zschokke,  Guillebeau 
and  Hess,  and  Degoix  have  shown  a  coccidium  which  is  18  to  25 
micra  long,  13  micra  wide,  in  the  stools  of  sick  animals  and  in  the 
gastro-intestinal  tract.  According  to  Guillebeau  these  coccidia  form 
four  spores  at  a  temperature  of  20°  to  30°  C.,  but  at  39°  C.  numerous 
small  round  spores  of  4  to  7  micra  diameter.  Young  cattle  can  be 
infected  artificially  with  these  coccidia  by  feeding  them,  and  after 
an  incubation  of  three  weeks  they  develop  typical  attacks  of  the 
disease.  The  coccidia  have  been  found  in  the  intestinal  epithelia, 
particularly  in  those  of  the  crypts  of  Lieberkiihn.  The  gastric 
mucosa  in  this  coccidial  infection  shows  inflammatory  and  hemor- 
rhagic  changes.  In  fatal  cases  all  the  organs  show  the  signs  of  anemia 
and  cachexia. 

Coccidiosis  in  sheep  has  been  reported  by  Rivolta,  Leuckart, 
Nocard,  Cooper,  Curtice,  Stiles,  McFadyean,  and  others.  The 
symptoms  are  similar  to  those  of  coccidiosis  in  cattle;  the  coccidia 
are  found  in  the  intestinal  epithelia,  but  they  have  not  been  found 
in  the  feces. 

Coccidium  tenellum,  claimed  to  be  the  cause  of  white  diarrhea  in 
chickens,  has  been  mentioned  in  Chapter  XXIV  under  the  head  of 
Bacterium  Pullorum. 

Coccidiosis  renalis  is  the  name  given  to  a  condition  in  which 
after  death  due  to  progressive  cachexia,  coccidia  have  been  found 
in  the  kidneys.  Ralliet  and  Lucet  have  reported  such  cases  in  geese, 
and  Paechinger  has  reported  one  case  in  a  horse  and  one  in  a  dog. 

A  skin  disease  of  hogs  known  as  hypotrichosis  localis  cystica, 
spiradenitis  coccidiosa,  "Schrotausschlag  der  Schweine"  (German), 
characterized  by  a  chronic  eruption  of  the  skin,  is  claimed  to  be  due 
to  the  Coccidium  fuscum  of  Alt,  which  invades  the  epithelia  of  the 
sebaceous  glands. 


572  HEMOSPORIDIA 


HEMOSPORIDIA. 

The  hemosporidia  include  some  of  the  most  important  pathogenic 
protozoa,  causing  disease  in  man  and  domestic  animals,  namely, 
the  plasmodium  which  causes  malaria  in  man  and  monkeys;  hemo- 
proteus,  the  cause  of  malaria  in  birds;  piroplasma,  or  Babesia,  the 
cause  of  piroplasmoses,  or  hemoglobinurias,  in  several  species  of 
domestic  animals.  To  the  piroplasmoses  also  belongs  Texas  fever  of 
cattle.  This  group  of  diseases  will  be  considered  separately  in  the 
next  chapter. 

Malaria. — Probably  no  other  disease  is  so  widely  prevalent  among 
human  beings  throughout  the  world,  except  in  the  most  northern 
and  southern  latitudes,  as  malaria.  It  is  spread  from  the  infected  to 
the  non-infected  by  mosquitoes  of  the  genus  anopheles. 

Malarial  parasites  were  first  seen  in  the  blood  of  patients  and 
considered  to  be  the  cause  of  the  disease  by  Laveran  in  1880.  Mar- 
ciafava  and  Celli  in  1885  gave  a  more  detailed  description  of  the 
microorganisms,  and  proposed  for  them  the  name  of  Plasmodium 
malariae,  under  the  erroneous  impression  that  they  were  dealing  with 
a  vegetable  microorganism.  This  name  is  still  retained,  though  the 
malarial  organisms  are  now  properly  classified  among  the  subphylum 
sporozoa,  order  hemosporidia.  Schaudinn  divides  the  species  into 
three  varieties,  namely,  Plasmodium  vivax  (the  parasite  of  tertian 
malaria),  Plasmodium  malaria  (the  cause  of  quartan  malaria),  and 
Plasmodium  falciparum  (the  organism  of  quotidian  or  estivo-autumnal 
malaria). 

The  malarial  fevers  are  characterized  by  a  very  definite  recurrence 
of  elevations  of  temperature  after  different  periods  of  time.  The 
fever  curve  may  rise  daily,  and  reach  its  maximum  at  an  almost  con- 
stant time  of  each  day;  this  is  the  so-called  quotidian  type.  Or  the 
apex  of  the  fever  curve,  generally  ushered  in  by  a  chill,  may  occur 
and  re-occur  after  forty-eight  hours;  this  is  the  tertian  type.  Or  it 
may  occur  always  after  seventy- two  hours,  this  is  the  quartan  type. 
These  febrile  and  afebrile  periods  depend  upon  the  natural  life 
history  of  different  varieties  of  malarial  parasites.  If  a  person  is 
infected  by  the  bite  of  a  mosquito  which  carries  the  parasites  as  the 
intermediate  host  the  plasmodia  get  into  that  person's  blood  and 
there  multiply.  For  a  certain  time  the  number  of  parasites  is  com- 
paratively small,  no  symptoms  develop,  and  the  patient  is  then  in 
the  period  of  incubation.  Then  an  outbreak  occurs,  characterized 
by  chills,  followed  by  fever.  This  is  repeated  after  twenty-four,  forty- 
eight,  or  seventy- two  hours,  according  to  the  variety  of  infecting 
plasmodium.  It  can  be  easily  shown  that  these  outbreaks  always 
occur  shortly  after  the  time  when  the  intracorpuscular  parasites 
break  up  into  merozoites,  or  asexually  produced  spores.  As  soon  as 
these  are  liberated  from  the  corpuscle  which  they  have  flestroyed 


PLATE  XII 


Life-cycle  of  Plasmodium  Vivax.     (After  Grassi  and  Sehaudinn.) 

The  hurtian  cycle  is  above  the  transverse  line,  somewhat  rearranged  by  Kisskalt  and  Hartmann. 
The  cycle  in  the  mosquito  is  beneath.  1  to  7,  schizogony;  1,  sporozoite;  2,  entrance  of  the  sporozoite; 
3  and  4,  growth  of  the  schizont;  5  and  6,  nuclear  division  of  the  schizont;  7,  formation  of  the 
merozoites;  8,  merozoites;  9a  to  12a,  growth  of  the  macrogametocyte;  96  to  126,  growth  of  the  micro- 
gametocyte;  13c  to  17c,  parthenogenesis  of  the  macrogametocyte;  13a  and  14a,  maturation  of  the 
macrogamete;  136  and  146,  growth  of  the  microgamete;  156,  microgamete;  16,  fructification;  17, 
ookinet;  18  to  20,  entrance  of  the  ookinet  into  the  stomach  wall  of  the  mosquito; 20  to  25,sporogony; 
22  and  23,  nuclear  multiplication  in  the  sporont;  24  and  25,  formation  of  the  sporozoites;  26,  passage 
of  the  sporozoites  to  the  salivary  gland ;  27,  salivary  gland  of  the  mosquito  with  sporozoites.  (Magni- 
fication, 4  to  17c,  1200  to  1;  18  to  27c,  600  to  1.) 


PLASMODIUM  VIVAX 


573 


they  invade  fresh  corpuscles,  and  either  directly  or  indirectly  are 
responsible  for  the  chill,  fever,  prostration,  etc. 

The  parasites  of  malaria  have  a  double  cycle  of  reproduction. 
Asexual  reproduction  (Schizogony)  by  spore  formation  (merozoites) 
occurs  in  the  blood  of  man  or  monkeys;  the  sexual  cycle  of  repro- 
duction by  copula  formation  of  sexually  differentiated  gametes  occurs 
in  the  body  of  the  intermediate  host,  the  mosquito  of  the  genus 
anopheles. 

Plasmodium  Vivax. — This  is  the  cause  of  tertian  malarial  fever. 
After  its  entrance  into  the  body  of  man  it  is  first  seen  in  the  interior 
of  red  blood  corpuscles  as  a  small,  quite  motile,  hyaline  body,  variable 

FIG.  200 


Anopheles  maculipennis:  adult  male  at  left,  female  at  right.     (Howard.) 

in  shape  on  account  of  the  contractility  and  ameboid  motion  of 
its  protoplasm.  After  a  time  the  latter  contains  reddish-brown, 
rather  fine,  diffusely  distributed  pigment,  which  increases  in  amount. 
While  the  perfectly  hyaline  bodies  are  difficult  to  see  under  the 
microscope  the  parasites  are  easily  visible  after  they  have  formed 
pigment,  particularly  as  the  granules  in  fresh  blood  are  in  constant 
motion  in  consequence  of  protoplasmic  contractility  and  currents. 
About  forty-eight  hours  after  its  first  entrance  into  the  red  blood 
corpuscle  the  plasmodium  has  reached  a  large  size,  and  now  fills  the 
enlarged  erythrocyte  almost  completely.  The  parasite  now  has  lost 
its  ameboid  motion,  is  round  in  shape,  and  divides  by  segmentation 
into  twelve  to  twenty-four  merozoites  (asexually  produced  spores). 


574  HEMOSPORIDIA 

The  merozoites,  which  are  from  1  to  3  micra  and  more  in  diameter, 
get  into  the  blood  plasma  and  from  there  infect  new  erythrocytes. 
The  early  differentiation  of  gametocytes  occurs  in  man,  but  the 
completion  of  this  change  only  takes  place  after  mosquitoes  have 
taken  up  the  parasite  with  the  blood.  The  gametocytes  then  become 
fully  developed,  form  gametes,  and  these  go  through  a  process  of 
maturation  in  the  intestinal  tract  of  anopheles.  Microgametes  then 
fertilize  the  macrogametes,  and  the  copula  formed  has  been  called 
ookinet  by  Schaudinn.  This  ookinet  penetrates  into  the  submucosa 
of  the  gut  of  the  mosquito,  and  grows  considerably  in  size.  After 
several  days  its  nucleus  divides,  the  cytoplasm  likewise  segments, 
and  the  naked  sporozoites  are  then  formed.  These  circulate  in  the 
body  of  the  mosquito,  and  many  of  them  finally  get  into  the  salivary 
glands  and  from  there  into  the  proboscis  of  the  biting  insect,  through 
which  they  are  ultimately  discharged  into  the  body  of  man.  Then  the 
asexual  cycle  starts  anew  until  merozoites  are  again  taken  up  by 
anopheles,  in  which  they  pass  through  the  sexual  part  of  the 
cycle. 

Plasmodium  Malarise. — The  parasite  causing  the  quartan  type  of 
malaria  is  like  the  preceding  one  first  seen  as  a  hyaline  body  in  the  red 
blood  corpuscle.    It  is,  however,  not  as  lively  motile  as  the  plasmodium 
vivax.     The  pigment  granules  which  subsequently  form  are  larger 
than  in  the  case  of  the  tertiary  parasite,  and  are  arranged  in  a  regular 
peripheral  and  not  in  a  diffuse  manner.    The  whole  parasite  remains 
smaller  and  the  infected  red  blood  corpuscle  does  not  become  abnor- 
mally large,  is  not  very  pale,  but  rather  of  a  dark  greenish  color. 
At  the  end  of  the  third  day  the  Plasmodium  malarise  is  full  grown, 
and  is  much  more  highly  refractive  than  the  Plasmodium  vivax. 
From  eight  to   twelve  merozoites  are  then  formed,  arranged  in  a 
regular  rosette.    The  merozoites  after  being  set  free  in  the  blood  plasma 
invade  new  corpuscles.    The  sexual  part  of  the  cycle  in  the  mosquito 
is  like  that  of  the  preceding  variety.    When  blood  containing  either 
the  Plasmodium  vivax  or  Plasmodium  malarise  is  obtained  by  the 
prick  of  a  needle  and  allowed  to  fall  on  a  slide,  covered  with  a  cover- 
glass,  protected  against  evaporation,  and  watched  under  the  micro- 
.scope,  the  formation  of  flagellated  forms  can  be  observed.     Fully 
grown  plasmodia  filled  with  very  actively  motile  pigment  form  wavy, 
slender  prolongations  of  the  protoplasm,  several  times  as  long  as  the 
diameter  of  the  main  body  of  the  parasite.     The  flagella  exhibit  a 
very  lively  whip-like  motion.    The  cells  which  have  undergone  this 
change  are  the  microgametocytes,  which  produce  the  microgametes  by 
the  breaking  loose  of  the  flagella  after  they  have  been  provided  with 
nuclear  substance  from  the  mother  cell  which  formed  them.     While 
the  formation  of  the  flagellated  organisms  occurs,  round  parasites, 
with  pigment  collected  in  larger  masses  in  a  peripheral  manner,  can 
be  seen.    These  plasmodia  are  the  macrogametocytes.    These  sexual 
forms,  which  frequently  can  be  seen  under  the  microscope  in  drawn 


PLASMODIUM  IMMACULATUM  OR  FALCIPARUM         575 

blood,  are  always  formed  in  the  gut  of  the  mosquito  as  the  first  step 
in  sexual  reproduction  as  outlined  above. 

Plasmodium  Immaculatum  or  Falciparum. — According  to  Marchiafava 
and  Bignani  and  Craig  this  occurs  in  two  varieties,  namely,  the 
quotidan  and  the  tertian.  These  are  described  by  Craig  as  follows: 
The  quotidian  parasite  after  invading  the  red  blood  corpuscles  is 
first  indistinct,  but  later  becomes  clear-cut  and  refractive.  There 
are  round  forms,  but  the  most  common  form  is  the  ring  form.  While 
most  observers  think  that  the  ring  form  is  only  apparent,  due  to  a 
very  thin  centre,  Craig  believes  that  this  type  of  plasmodium  gen- 
erally forms  real  rings  in  the  interior  of  the  erythrocytes.  Schaudinn 
believes  that  the  ring  form  is  due  to  a  large  vacuole  in  the  centre 
of  the  organism.  The  ameboid  motion  of  the  rings  is  very  active; 
the  infected  red  blood  corpuscles  are  frequently  undersized,  and 
they  may  be  crenated.  One  corpuscle  may  contain  two  or  three 
rings.  The  Plasmodium  falciparum  assumes  the  shape  which  has 
been  likened  to  a  signet  ring.  This  form  is  brought  about  by  a 
collection  of  most  of  the  protoplasm  in  one  point,  while  the  remainder 
is  arranged  as  a  thin  circular  strip.  The  pigment  first  appears  in 
the  thickest  portion  of  the  signet  ring.  These  formations  are  more 
common  in  the  tertian  than  in  the  quotidian  type.  The  pigment  is 
rather  scanty,  but  very  dark  in  color,  and  collected  somewhere  at 
the  edge  of  the  parasite.  Sometimes  the  pigment  consists  of  a  very 
few  distinct  granules  only.  Segmenting  forms  are  rarely  seen  in 
the  peripheral  blood,  but  they  are  common  in  the  spleen,,  from  which 
they  may  be  obtained  by  puncture.  At  the  time  of  segmentation  the 
pigment  becomes  collected  at  the  centre.  There  are  six  to  eight 
very  small  round  or  oval  merozoites  formed.  In  the  circulating 
blood  Plasmodium  falciparum  forms  the  crescents,  which  are  so  char- 
acteristic for  this  variety  of  malarial  parasite.  These  crescents  are 
curved.  Sometimes  they  fill  the  greater  part  of  a  red  blood  corpuscle 
or  even  protrude  out  of  it  at  one  or  both  ends,  and  they  may  finally 
show  a  remnant  of  the  corpuscle  as  a  cap  lying  in  the  concavity. 
The  pigment  is  found  in  the  centre  of  the  crescent,  where  it  is  often 
arranged  in  a  regularly  circular  manner.  The  crescents  are  the  macro- 
gametocytes  of  the  plasmodium  falciparum,  and  a  number  of  observers 
have  seen  a  binary  division  of  the  crescents  in  the  infected  blood. 

The  tertian  subvariety  of  the  Plasmodium  falciparum  in  its  early 
stage  after  invasion  of  the  blood  corpuscles  is  larger  than  the  quartan 
subvariety,  very  highly  refractive,  and  the  signet-ring  forms  are  still 
more  marked.  Ameboid  motion  is  less  rapid.  The  hyaline  forms 
become  pigmented  in  twenty  to  twenty-four  hours;  the  abundant 
pigment  consists  of  very  fine  reddish-brown  granules,  which  are 
generally  motile.  As  growth  progresses  the  ameboid  motion  is  lost. 
Segmentation  occurs  after  forty-eight  hours,  and  the  organism  then 
occupies  about  one-half  of  the  infected  cell.  From  ten  to  fifteen 
merozoites  are  generally  formed,  sometimes  as  many  as  twenty- 


576  HEMOSPORIDIA 

four.  Segmentation  is  generally  not  seen  in  the  peripheral  blood, 
but  it  can  be  shown  in  blood  drawn  by  puncture  from  the  spleen. 

Examination  of  Blood. — Examination  of  the  blood  for  the  plas- 
modium  of  malaria  is  made  on  unstained  fresh  and  on  dried  specimens. 
The  latter  are  best  stained  with  the  Romanowski  stain  or  one  of  its 
modifications,  such  as  the  Wright  stain. 

Hemosporidia  in  Birds. — Birds  frequently  harbor  trypanosomes  in 
their  blood,  as  has  been  stated  previously.  Two  kinds  of  hemosporidia 
have  also  been  found. 

Proteosoma,  or  Cytosporon  danilewsky,  or  Hemameba  relic ta  is  found 
in  birds  of  the  sparrow  family,  in  predatory  birds,  pigeons,  crows, 
etc.  The  life  cycle  of  this  hemosporidium  is  described  by  Ruge 
as  follows:  The  youngest  parasites  are  seen  in  the  erythrocytes  of 
birds  as  a  small,  round,  refractive,  sharply  defined  body,  with  one 
minute  pigment  granule.  The  young  proteosoma  is  generally  situated 
near  one  pole  of  the  blood  corpuscle,  or  it  may  be  near  its  nucleus. 
The  parasite  is  not  motile;  it  grows  rapidly  and  causes  the  nucleus 
of  the  erythrocyte  to  move  or  turn  away  from  it.  During  growth  the 
pigment  increases  and  becomes  lumped  together.  Afterward  the 
organism  breaks  up  into  six  to  eight  merozoites,  which  are  arranged 
as  a  rosette  or  in  fan-shape.  The  largest  forms  break  up  into  twelve  to 
fifteen  spores.  The  corpuscles  infected  with  the  dividing  parasites 
lose  their  regular  shape  and  burst.  The  free  spores  then  invade 
fresh  blood  corpuscles.  Sexually  differentiated  gametes,  however, 
are  also  formed  in  the  blood  of  infected  birds.  Their  further  develop- 
ment occurs  in  the  intermediary  host,  a  mosquito  (Culex  pipiens). 
The  macrogametes  become  large  and  round;  the  flagellated  micro- 
gametes  are  formed  in  a  manner  resembling  the  formation  which 
occurs  in  anopheles  in  the  case  of  the  human  malarial  plasmodia. 
The  ookinetes  are  formed  in  the  stomach  of  the  mosquito  about 
twelve  hours  after  it  has  taken  up  the  infected  blood.  Seven  to  ten 
days  later  the  sickle-shaped  sporozoites  are  found  in  the  salivary 
glands  of  the  insect.  Sporozoite  development,  however,  occurs  for 
a  short  time  only,  during  temperatures  of  24°  to  30°  C.;  between  15° 
to  23°  C.  their  development  is  much  retarded,  and  at  lower  temper- 
atures it  ceases  entirely. 

Halteridium,  or  hemoproteus,  infects  frequently  the  red  blood  cor- 
puscles of  predatory  birds,  singing  birds,  and  particularly  pigeons. 
In  tropical  and  subtropical  countries,  R.  Koch  found  pigeons  very 
generally  infected.  Halteridium  is  generally  seen  in  its  typical  dumb- 
bell shape  in  close  apposition  with  the  nucleus  of  the  erythrocyte. 
The  dumb-bell-shaped  parasites,  according  to  .Ruge,  occur  in  birds 
in  two  types,  a  hyaline  form  representing  the  male,  and  a  finely 
granular  form  representing  the  female  element.  MacCallum  has  ob- 
served how  the  microgametocytes  of  the  halteridium  become  flagel- 
lated and  how  the  microgametes  penetrate  into  the  macrogametes. 
The  life  cycle  of  halteridium,  however,  is  not  yet  completely  known. 


NOSEMA  BOBYCIS  577 

Schaudinn  has  claimed,  as  previously  stated,  that  Hemoproteus  noctuse 
(the  parasite  of  the  owl)  develops  in  the  body  of  Culex  pipiens  into  a 
flagellate,  a  trypanosome,  or  allied  organism;  but  Novy  and  his 
co-workers  maintain  that  the  flagellates  seen  by  Schaudinn  in  culex  are 
not  a  phase  in  the  life  cycle  of  hemoproteus  but  simply  a  parasitic 
flagellate  of  the  insect. 

FIG.  201 


Culex  pipiens;   adult  female.     (Howard.) 


MICROSPORIDIA. 

Nosema  Bobycis. — Nosema,  or  Glugea  bombycis,  is  the  most  widely 
known  microsporidium.  It  is  the  cause  of  the  silkworm  disease, 
pebrine  (French),  studied  by  Pasteur.  During  the  years  1854  to  1867 
this  microorganism  is  estimated  to  have  caused  losses  in  France 
amounting  to  about  $200,000,000.  The  silkworm  affection  caused 
by  this  microsporidium  was  the  first  disease  studied  by  Pasteur,  and 
to  a  large  extent  conquered  by  his  prophylactic  measures.  These 
studies  initiated  him  into  the  field  of  preventive  medicine,  where  he 
later  gained  such  immortal  fame.  Nosema  bombycis,  therefore,  is  a 
pathogenic  organism  of  great  historical  interest.  It  is  believed  that 
the  caterpillar  of  the  silk  moth  (Bombyx  mori)  infects  itself  with  its 
food  with  the  parasites.  The  caterpillars,  extensively  infected,  die 
before  they  have  had  an  opportunity  to  form  chrysalides  inclosed  in 
cocoons  of  silk.  Those  less  infected  can  go  on  to  full  development 
37 


578 


SARCOSPORIDIA 


as  male  and  female  moths.  Since  the  sexual  organs  are  infected  with 
the  parasites  they  transfer  the  infection  to  the  ova  and  from  these 
to  the  young  caterpillars.  In  this  manner  the  infection  may  be  con- 
tinued from  generation  to  generation,  bringing  about  both  great 
mortality  and  an  inferior  quality  of  cocoons.  Pasteur  showed  how 
to  distinguish  microscopically  the  infected  from  the  non-infected 
ova,  and  in  this  manner  enabled  the  breeders  of  silkworms  to  weed 
out  the  disease. 

FIG.  202 


Nosema  bombycis:  1  to  5,  spore  formation;  6,  infected  follicle  of  testicle;  7,  spores;  a,  b, 
fresh;  c,  d,  treated  with  nitric  acid.  The  acid  causes  them  to  swell  up  and  increase  in  size  by 
at  least  a  half,  at  the  same  time  making  the  polar  capsule  distinct.  In  d,  the  filament  is 
extruded.  (After  Balbiani.) 

SARCOSPORIDIA. 

Sarcosporidia  are  protozoan  parasites  occurring  in  the  muscle 
fibers  of  a  large  variety  of  animals,  such  as  hogs,  sheep,  cattle,  horses, 
dogs,  cats,  rabbits,  rats,  mice,  monkeys,  chickens,  and  some  other 
domestic  and  wild  birds.  They  have  also  been  occasionally  found  in 
the  muscles  of  man.  In  the  muscles  of  affected  animals  sarcosporidia 
form  elongated  sausage-like  spore  sacs,  which  have  been  known  for 
a  long  time  as  Miescher's  or  Rainey's  tubules.  These  spore  sacs 
generally  measure  from  ^  to  4  mm.  in  length,  but  there  is  a  sarco- 
sporidium  (Balbiana  gigantea)  found  in  the  esophagus  of  sheep 
which  may  attain  the  size  of  a  hazelnut.  Older  spore  sacs  sometimes 
show  a  double  membrane,  the  outer  one  exhibiting  a  radial  striation, 
as  if  it  were  provided  with  short,  rod-like  cilia.  The  real  character 
of  this  structure  has  not  yet  been  clearly  made  out;  it  is  now  more 
generally  believed  that  the  striae  represent  fine  pore-canaliculi.  The 
interior  of  the  sac  is  divided  into  compartments  by  fine  partition 
walls  arising  from  the  inner  membrane.  Included  in  these  chambers 
are  the  sporoblasts  and  their  spores.  In  the  entoplasm  of  the  smallest 
sacs,  balls  4  to  5  micra  in  diameter,  which  show  an  indistinct  nucleus, 


PLATE   XIII 


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Sareosporidia  in  Muscle  of  Cattle. 


SARCOSPORIDIA  579 

are  seen.  The  latter  subsequently  divides  the  cytoplasm  segments, 
and  in  this  manner  the  balls  become  sporoblasts  and  their  contents 
are  transformed  into  numerous  crowded,  curved,  oval,  sickle-  or 
crescent-shaped  spores.  It  is  not  known  how  the  sarcosporidia  first 
gain  entrance  into  animals,  nor  is  their  life  cycle  well  known.  It  is 
believed  that  part  of  it  occurs  in  an  intermediate  host,  and  it  has  been 
claimed  that  mollusks  (snails)  play  this  role.  Sarcosporidium  infection 
in  animals  is,  as  a  rule,  a  harmless  process.  The  parasites,  however, 
may  be  very  numerous  throughout  the  entire  muscular  system,  and 
may  so  interfere  with  its  nutrition  and  function;  or  the  sacs  may  be 
in  locations  where  they  may  do  harm.  The  giant  sarcosporidia  in 
the  esophagus  of  the  sheep,  for  example,  may  interfere  with  deglu- 
tition and  respiration.  A  few  cases  of  sarcosporidia  infection  of 
the  laryngeal  muscles  of  horses  have  been  reported;  the  author  has 
seen  such  a  case  in  which  the  larynx  became  the  seat  of  considerable 
inflammatory  infiltration,  with  respiratory  disturbances.  Sarco- 
sporidia can  be  studied  from  fresh  material,  unstained  and  also  in 
stained  sections  of  infected  muscles. 

Sarcocystis  miescheriana  is  the  sarcosporidium  most  commonly 
found  in  hogs.  The  sacs  are  from  0.5  to  4  mm.  long  and  3  mm.  wide. 
The  pansporoblasts  (the  balls  which  subsequently  develop  the  spores 
in  their  interior)  are  5  to  6  micra  in  diameter.  If  pork  is  extensively 
infected  by  these  sarcosporidia  it  is  "off  color,"  yellowish  or  grayish 
red.  The  sacs  often  show  the  evidences  of  leukocytic  infiltration 
and  of  calcareous  degeneration. 

Sarcocystis  bertrami,  generally  9  to  12  mm.  long,  is  found  in  the 
muscles  of  the  horse.  It  may  cause  interstitial  myositis,  and  may 
become  dangerous  when  located  in  the  muscles  of  the  larynx.  It 
sometimes  affects  the  muscles  of  the  hind  leg  of  young  horses  and 
causes  lameness. 

Sarcocystis  tenella  infects  the  muscles  of  sheep  and  goats.  The 
organism  varies  considerably  in  length,  namely,  from  40  micra  to 
2  cm.  The  sac  membrane  is  very  delicate  in  young  parasites,  but 
becomes  thick  and  tough  when  they  grow  older.  The  large  cysts 
contain  a  layer  of  sporoblasts  along  the  interior  of  the  membrane, 
but  the  centre  is  composed  of  an  empty  meshwork  only.  The  spores 
are  first  perfectly  round,  but  small  and  kidney-shaped  after  their 
full  development.  This  sarcosporidium  invades  many  of  the  muscles 
of  sheep  and  goats,  and  also  the  heart  muscle  and  its  endocardium. 

Sarcocystis  lindemann  has  been  found  by  R.  Koch  and  by  Kartulis 
in  Africa  in  the  muscles  of  man  and  also  by  others  a  few  times  in 
other  parts  of  the  world. 

Balbiana  rileyi,  another  sarcosporidium,  was  found  by  Stiles  in 
the  muscles  of  ducks  in  the  United  States. 


580  SARCOSPORIDIA 


QUESTIONS 

1.  What  are  the  most  important  common  characteristics  of  the  protozoan 
subphylum  sporozoa? 

2.  Give  a  definition  of  the  order  of  coccidia. 

3.  Give  a  definition  of  the  order  of  hemosporidia. 

4.  Give  a  definition  of  the  order  of  microsporidia. 

5.  Give  a  definition  of  the  order  of  sarcosporidia. 

6.  In  what  kind  of  animals  and  in  what  tissues  are  coccidia  found  as  patho- 
genic parasites? 

7.  Describe  the  cytoplasm  and  the  nucleus  of  typical  coccidia. 

8.  Describe  the  asexual  and  sexual  life  cycle  of  a  coccidium. 

9.  What  is  meant  by  polar  bodies?    WTiat  by  the  process  of  maturation? 

10.  Explain  the  terms  macrogametes  and  microgametes  and  copula. 

11.  Describe  the  most  important  pathologic  changes  and  the  symptoms  of 
coccidiosis  in  rabbits. 

12.  What  is  the  other  name  of  Coccidium  cuniculi?    Describe  its  morphology. 

13.  Describe  coccidiosis  in  cattle. 

14.  What  other  domestic  animals  may  suffer  from  coccidiosis? 

15.  What  is  hypotrichosis  localis  cystica  of  hogs?    What  causes  it? 

16.  Name  some  hemosporidia  causing  important  diseases  in  man  and  animals. 

17.  What  kind  of  a  disease  is  malaria?    How  is  it  spread  from  the  infected 
to  the  non-infected? 

18.  Describe  the  Plasmodium  vivax  in  the  blood  of  man. 

19.  Describe  its  sexual  cycle  in  the  anopheles  mosquito. 

20.  What  is  an  ookinet? 

21.  Describe  the  Plasmodium  malarise. 

22.  Describe  the  Plasmodium  falciparum. 

23.  What  is  the  cause  of  the  regularity  of  the  fever  curve  in  the  various  forms 
of  malaria? 

24.  Name  the  hemosporidia  in  birds. 

25.  What  other  protozoa  infect  the  blood  of  birds? 

26.  Describe  proteosoma. 

27.  Describe  halteridium  or  hemoproteus. 

28.  Has  this  hemosporidium  a  flagellate  stage  in  its  life  cycle? 

29.  What  kind  of  an  organism  is  Nosema  or  Glugea  bombycis? 

30.  Describe  the  morphology  of  sarcosporidia  and  state  where  they  are  found. 
Are  they  very  pathogenic? 


CHAPTEK    LIV. 

PIROPLASMA  BOVIS— TEXAS  FEVER  AND  PIROPLASMOSES  IN 
OTHER  ANIMALS. 

PIROPLASMA   BOVIS. 

Occurrence  and  Historical. — Texas  fever  is  a  disease  of  cattle  due 
to  a  protozoan  microorganism  infecting  the  blood  plasma  and  the 
red  blood  corpuscles,  and  now  generally  known  as  Piroplasma  bigem- 
inum,  or  Babesia  bigemina.  The  disease  has  been  and  is  known 
under  a  variety  of  names,  such  as  splenic  fever  (this  name,  however, 
is  now  more  commonly  used  for  anthrax),  Spanish  fever,  Mexican 
fever,  Southern  cattle  fever,  Australian  tick  fever,  Tristeza,  red  water, 
black  water,  hemoglobinuria  of  cattle,  paludism  of  cattle,  piroplas- 
mosis  of  cattle,  etc.  The  disease  has  undoubtedly  existed  in  the  old 
world,  where  it  was  formerly  known  as  wood  and  moor  ill,  for  a  long 
time.  It  first  attracted  attention  both  in  Europe  and  America  about 
the  middle  of  the  last  century.  At  this  time  the  disease  was  studied 
by  veterinarians  in  Russia  and  France,  and  also  became  the  subject 
of  much  inquiry  in  this  country,  when  cattle  coming  from  Texas 
introduced  the  disease  into  Indiana  and  Illinois,  where  its  ravages 
became  alarming,  and  when  it  likewise  appeared  in  cattle  brought  from 
the  West  to  the  slaughtering  houses  of  New  York.  A  commission 
appointed  in  the  latter  State  studied  the  disease  and  issued  a  report 
in  1868.  It  described  the  symptomatology  and  pathology  of  the 
disease  correctly,  but  did  not  ascertain  its  cause.  Later  investigators 
accused  various  bacteria  of  being  the  cause  of  the  disease,  but  erro- 
neously, as  was  subsequently  shown.  In  1888  Babes  studied  the  dis- 
ease in  Roumania,  and  reported  that  he  had  discovered  in  the  interior 
of  the  red  blood  corpuscles  of  animals  sick  with  hemoglobinuria 
diplococci-like  bodies  which  could  be  stained  with  methylene  blue, 
but  which  could  be  cultivated  only  with  difficulty.  Babes  thought 
that  these  diplococci-like  bodies  were  neither  bacteria  nor  protozoa, 
but  some  organism  intermediate  between  them.  While  Babes  un- 
doubtedly saw  and  correctly  described  the  organisms  causing  Texas 
fever  in  cattle,  he  was  in  error  concerning  their  alleged  cultural 
properties,  and  had  no  correct  conception  of  their  mode  of  entrance 
into  the  body  of  the  infected  animal,  which  he  thought  was  through 
the  drinking  water.  The  etiology  of  Texas  fever  was  cleared  up 
completely  in  1893  by  Theobald  Smith  and  Kilbourne.  They  saw 
the  infecting  protozoa,  described  them  correctly,  and  showed  that  the 


582  PIROPLASMA  BOVIS 

mode  of  infecting  cattle  was  by  transmission  through  biting  and  blood- 
sucking ticks.  Their  work  was  afterward  confirmed  in  other  parts 
of  the  world  in  the  observation  of  identical  or  at  least  similar  blood 
infections  of  cattle  in  Finland,  Italy,  Australia,  Africa,  Germany, 
and  South  America.  Smith  and  Kilbourne  first  named  the  blood 
parasite  found  in  Texas  fever  pirosoma;  later  the  organism  was  called 
apiosoma.  Since,  however,  these  family  names  had  previously  been 
applied  to  other  organisms,  the  name  was  subsequently  changed  to 
Piroplasma  bigeminum,  which  means  pear-shaped  protoplasmic 
twin  bodies. 

Pathologic  Anatomy. — If  the  disease  has  taken  a  very  rapid  course 
the  carcass  may  be  full  and  rounded;  if  the  animal  has  been  sick 
for  a  number  of  days  there  is  generally  emaciation  and  evidence 
of  rapid  loss  of  weight.  In  fat  cattle  which  have  contracted  the 
disease  and  died  from  it  during  or  shortly  after  transit  a  deep  orange 
hue  of  the  subcutaneous  and  other  connective  tissues  is  one  of  the 
most  characteristic  postmortem  findings;  frequently  the  muscles 
show  a  deep  mahogany  yellow  tint.  In  thin  milch  cows  and  Southern 
stock  cattle  the  icteric  discolorization  of  the  tissues  is  often  absent. 
The  degree  of  icteric  discolorization  of  the  tissues  depends  upon  the 
number  of  red  blood  corpuscles  which  have  been  destroyed  by  the 
infecting  parasites  and  the  amount  of  hemoglobin  which  has  first 
gone  into  solution  in  the  blood  plasma  and  has  subsequently  been 
deposited  in  the  tissues  or  excreted  by  the  urine.  Ticks  are  often 
found  adhering  to  the  skin  of  an  animal  dead  from  Texas  fever,  and 
here  and  in  places  where  they  have  fallen  off  edematous  and  hemor- 
rhagic  patches  are  seen. 

Microscopic  examination  of  the  blood  shows  a  more  or  less 
marked  diminution  of  the  number  of  red  blood  corpuscles  (oligo- 
cythemia).  The  decrease  in  red  blood  corpuscles  from  a  normal  of 
about  7,000,000  may  be  down  to  2,000,000  or  even  much  less.  If 
properly  studied  and  examined  with  a  high  power,  numerous  red  blood 
corpuscles  show  the  specific  cause  of  the  disease,  the  piroplasma. 

The  heart  often  shows  subpericardial  and  subendocardial  petechiae 
and  ecchymoses;  occasionally  the  myocardium  shows  cloudy  swelling 
and  fatty  degeneration.  The  lungs  are  at  times  somewhat  congested, 
and  may  likewise  show  small  hemorrhagic  spots.  The  peritoneal 
cavity  may  show  a  slight  amount  of  yellowish  serum.  The  spleen  is 
very  much  enlarged,  of  a  dark  brown  color,  and  the  pulp  very  soft. 
The  liver  is  enlarged,  and  shows  a  mottled  appearance  on  the  cut 
surface;  the  centres  of  the  lobules  are  yellowish,  the  periphery  reddish. 
Frequently  the  whole  liver  shows  an  icteric  color.  The  bile  ducts 
are  much  congested  with  thickened  bile  and  stand  out  as  markedly 
as  if  they  had  been  artificially  injected.  The  gall-bladder  is  distended 
and  filled  with  thickened  bile,  the  appearance  of  which  has  been 
likened  to  masticated  grass.  The  kidneys  show  hemorrhagic  and 
edematous  congestion  and  parenchymatous  degeneration  with  widen- 


MORPHOLOGY  OF  THE  ORGANISM 


583 


FIG.  203 


ing  of  the  cortical  portion  and  cloudy  swelling  of  the  epithelia.  Small 
hemorrhagic  spots  are  often  seen  in  the  cortical  and  medullary  por- 
tions and  also  in  the  mucosa  of  the  renal  pelvis.  Petechise  and 
ecchymoses  are  also  found  in  the  gastric  mucosa,  while  the  small 
intestines  show  congestion  generally  without  hemorrhages,  but  the 
cecum  and  colon  frequently  show  hemorrhages  and  are  often  of  a  deep 
red  or  purplish-brown  color.  The  urinary  bladder  often  contains  much 
hemoglobin-stained  urine,  which  may  contain  so  much  of  the  blood- 
coloring  matter  that  it  is  of  a  port-wine  color. 

Microscopic  examination  of  sections  of  the  various  organs  shows 
the  presence  of  numerous  piroplasmata  in  the  capillaries. 

Diagnosis. -The  diagnosis  of  the  disease  in  typical  acute  cases 
is  generally  comparatively  easy  on  account  of  the  characteristic 
symptoms,  including  the  bloody  urine  (hemoglobinuria),  and  it  can 
be  established  beyond  any  doubt 
by  a  microscopic  examination  of 
stained  blood  specimens.  These 
are  best  prepared  in  the  follow- 
ing manner: 

1.  Clean    an    ear  of    the    sick 
animal  well  with  water  and  then 
with  alcohol  and  dry  it  after  the 
cleansing. 

2.  Make  a  small  incision,  with 
a  scalpel,  small  pair  of  scissors, 
or  with  a  large,  sharp,  triangular 
pointed  needle. 

3.  Allow  a   drop  of  blood  to 
flow  on  a  glass  slide,  previously 
well  cleaned  with  alcohol  (so  that 
there  is  no  greasy  matter  on  it). 

4.  With  the  margin  of  one  end   Smear  from  the  kidneys  of  an  animal  dead 

Of  another  clean  slide    Spread  OUt    from    the   disease.      Hematoxylin-eosin    stain. 

the  drop  of  blood  in  a  thin,  even    x  100°-   <Author's  preparation.) 
layer  on  the  first  slide. 

5.  Allow  the  slide  to  become  air  dry  as  quickly  as  possible.    Drying 
may  be  hastened  by  waving  the  moist  slide  rapidly  in  the  air. 

6.  Fix  the  blood  film  on  the  slide.    The  best  method  is  to  immerse 
it  in  absolute  alcohol  for  twenty-five  minutes  or  more. 

7.  Stain  with  Loeffler's  blue.     If  instead  of  Loeffler's  methylene 
blue   a   Romanowski   stain   or   one   of   its   modifications    (Wright's 
stain)  is  to  be  used,  it  is  best  either  not  to  fix  the  dry  blood  film  at 
all  or  to  fix  it  in  pure  methyl  alcohol,  which  is  the  solvent  of  the  stains 
of  the  Romanowski  type. 

Morphology   of    the    Organism. — The    Piroplasma   bigeminum,   or 
Babesia1  bigemina,  can,  according  to  Smith  and  Kilbourne,  be  seen 

i  In  honor  of  Babes,  who  first  saw  them. 


Piroplasmosis     of     cattle      (Texas     fever). 


584  PIROPLASMA  BOVIS 

in  the  fresh  blood  of  cattle  suffering  from  Texas  fever  as  a  pair  of 
small,  pale  bodies,  each  one  pear-shaped  and  touching  the  other,  or 
directed  toward  each  other  with  their  narrow-pointed  ends.  They 
vary  in  size  in  different  blood  corpuscles,  but  the  two  forming  a  pair 
are  generally  fairly  equal.  They  are  about  2  to  4  micra  long  and  1.5 
to  2  micra  wide  at  the  broader  ends.  The  pointed  ends  touch  or 
nearly  touch  each  other  and  the  axes  of  the  two  bodies  form  a  varying 
angle;  they  may  be  almost  parallel  or  they  may  form  a  straight  line, 
and  they  may  show  any  intermediate  stage  between  these  two  extremes. 
The  piroplasmata  are  of  a  homogeneous  not  granular  appearance, 
and  they  are  well  differentiated  from  the  stroma  of  the  red  blood 
corpuscles  in  which  they  are  found.  The  smaller  forms  are  generally 
perfectly  homogeneous,  the  larger  pear-shaped  bodies  often  show  at 
the  periphery  of  the  large  end  a  small,  highly  refractive,  somewhat 
darker  round  body  of  0.1  to  0.2  micron  in  diameter,  and  also  at  or 
near  the  centre  of  the  large  end  a  round  or  oval  body  of  0.5  to  1  micron 
in  diameter.  Sometimes  the  piroplasmata  in  fresh  blood  show  ameboid 
motion  with  a  change  in  the  contour  of  their  body,  but  without  the 
formation  of  any  well-marked  pseudopodia.  Dead  or  dying  organisms 
lose  the  pear  shape  and  assume  a  round  outline.  If  stained  cover- 
glass  or  slide  preparations  are  made  from  blood  containing  piro- 
plasmata the  alkaline  anilin  stain  is  seen  to  have  been  taken 
generally  more  intensely  at  the  margin  than  in  the  interior.  Not  all 
parasites  show  the  pear  shape  or  occur  in  pairs;  many  are  single, 
round,  oval,  or  irregular. 

According  to  Smith  and  Kilbourne,  usually  only  1  per  cent,  of 
the  erythrocytes  are  infected,  but  shortly  before  death  the  number 
of  infected  corpuscles  may  be  from  5  to  10  per  cent.  As  the  fever 
disappears  the  parasites  likewise  disappear  from  the  blood.  If 
the  animal  dies  the  blood  in  the  capillaries  of  the  internal  organs 
often  shows  an  enormous  infection.  The  piroplasmata  are  most 
numerous  in  the  kidneys  (in  50  to  80  per  cent,  of  the  red  blood  cor- 
puscles), and  after  them  in  the  liver  and  spleen,  and  they  are  also 
found  free  in  these  organs  between  the  red  corpuscles.  A  few  hours 
after  the  death  of  their  host  the  parasites  evidently  in  consequence 
of  degenerative  changes  lose  the  pear  shape  and  assume  a  round 
form.  These  degenerative  or  involution  forms,  however,  are  probably 
not  dead  parasites,  since  such  blood  retains  its  infective  character, 
and  since  blood  infected  with  piroplasma  and  obtained  under  aseptic 
conditions  may  remain  virulent  after  a  stay  of  sixty  days  in  the 
refrigerator.  In  the  incubator  at  37°  C.  such  blood  remains  virulent 
for  only  about  one  week.  Smith  and  Kilbourne  have  also  described 
coccus-like  bodies,  measuring  from  0.2  to  0.5  micron,  in  5  to  50  per 
cent,  of  the  red  blood  corpuscles  in  the  mild  autumnal  form  of  the 
disease  in  Texas  cattle.  These  bodies  can  generally  not  be  seen 
unstained,  but  only  after  treatment  with  methylene-blue  solution. 
They  are  looked  upon  by  the  investigators  named  as  a  form  in  the 


ANIMALS  SUSCEPTIBLE  585 

life  cycle  of  the  piroplasma,  not  as  degenerative  basophilic  granules 
of  the  protoplasm  of  the  red  blood  corpuscles. 

Kossel  and  Weber  studied  the  hemoglobinuria  of  cattle  in  Finland 
and  confirmed  the  previous  observations  of  Smith  and  Kilbourne, 
and  were  able,  by  the  use  of  the  Romanowski  stain,  to  add  further 
morphologic  details.  They  found  that  the  very  smallest  intra- 
corpuscular  parasites,  which  have  about  one-sixth  of  the  diameter  of 
the  red  blood  corpuscles,  are  very  delicate  ring  bodies,  the  margin 
of  which  stains  red,  the  inner  portions  blue.  Other  small  parasites 
are  irregular  in  shape  and  show  the  beginning  of  an  arrangement 
of  the  chromatic  substance  into  two  portions.  In  somewhat  larger 
bodies  the  division  of  the  chromatin  into  two  parts  has  become  quite 
distinct,  while  sometimes  the  chromatin  is  split  up  into  four  portions. 
These  authors,  however,  were  not  able  to  demonstrate  in  piroplasma, 
asexually  or  sexually,  dividing  bodies  as  they  occur  in  human  and 
avian  malarial  parasites  which  form  spores  by  two  distinct  types. 

While  nothing  definite  is  known  as  to  the  propagation  of  the  piro- 
plasma in  the  body  of  infected  cattle  it  is  quite  evident  that  it  must 
take  place  in  some  way,  since  an  enormous  increase  in  the  number  of 
parasites  within  a  short  time  can  be  observed  in  certain  stages  of  the 
disease.  Doflein  has  observed  that  the  nucleus-like  body  in  the 
interior  of  the  parasites  under  some  conditions  breaks  up  into  three, 
four,  or  more  smaller  fragments.  He  looks  upon  this  process  as  an 
asexual  spore-formation  (schizogony),  and  he  considers  the  large  pear- 
shaped  bodies  as  sexual  forms  (gametocytes). 

Whether  the  piroplasmata  found  in  animals  of  the  cattle  tribe  in 
various  parts  of  the  world  are  one  identical  species,  or  whether  they 
are  varieties  or  distinctly  different  species,  is  a  question  which  cannot 
as  yet  be  decided  definitely.  In  East  Africa,  Robert  Koch  found 
bacilli-like  forms,  often  four  in  one  corpuscle,  in  a  very  large  percentage 
of  the  red  blood  corpuscles  in  the  fatal  hemoglobinuria  of  cattle. 
These  rods  by  curving  upon  themselves  formed  ring-like  bodies; 
they  were  generally  thicker  in  the  middle  than  at  either  end,  and  by 
intermediate  forms  gradually  lead  to  the  typical  shape  of  the  piro- 
plasma. Since  such  forms  have  never  been  seen  in  Texas  fever  in  the 
United  States,  this  East  African  piroplasmosis  is  perhaps  due  to  a 
species  different  from  Piroplasma  bigeminum. 

Animals  Susceptible. — The  piroplasmosis  of  cattle  in  America  is 
not  transmissible  to  other  animals.  Experiments  have  been  made 
upon  horses,  asses,  sheep,  rabbits,  guinea-pigs,  dogs,  cats,  pigs,  mice, 
rats,  and  chickens.  The  tests  were  all  negative.  If,  however,  blood 
from  an  animal  sick  with  Texas  fever  is  inoculated  by  any  one  of  a 
variety  of  methods,  such  as  intravenous,  subcutaneous,  intraperitoneal, 
intramuscular,  intracerebral,  into  a  healthy  head  of  cattle  a  marked 
elevation  of  the  temperature  takes  place,  after  three  to  seven  days, 
and  piroplasmata  may  be  seen  in  the  circulating  blood.  After  a 
few  more  days  the  number  of  red  blood  corpuscles  and  the  hemoglobin 


586  PIROPLASMA  BOVIS 

become  diminished  and  the  urine  in  grave  cases  assumes  a  dark  color. 
The  best  method  for  making  the  inoculation  experiments  is  to  use 
5  to  10  c.c.  of  defibrinated  blood  from  an  infected  animal.1 

Animals  infected  with  such  blood  may  acquire  a  fatal  infection,  or 
the  infection  may  take  a  moderately  severe  or  even  a  mild  course. 

Natural  Mode  of  Transmission. — Texas  fever,  in  the  natural  course 
of  events,  always  is  transmitted  from  an  infected  animal  to  a  healthy 
one  by  blood-sucking  ticks.  The  tick  which  acts  as  the  intermediate 
host  of  the  Piroplasma  bigeminum  in  this  country  is  called  Boophilus 
bovis,  Rhipicephalus  annulatus,  or  Margaropus  annulatus.  In 
northern  Europe  piroplasmosis  of  cattle  is  spread  by  the  tick,  known 
as  Ixodes  reductus.  It  is  claimed  that  this  tick  also  occurs  in  America, 
and  may  here  be  concerned  in  the  spreading  of  Texas  fever.  The 
biting  and  sucking  ticks  probably  discharge  with  their  saliva  an  irri- 
tating fluid  which  produces  the  local  hyperemia  and  which  also 
introduces  the  protozoan  parasites  into  the  bitten  animal.  This 
discharge  of  piroplasma  from  the  intermediary  host  (the  tick)  to 
cattle  is  similar  to  that  of  the  Plasmodium  malaria?  by  mosquitoes 
(anopheles)  into  man.  The  developmental  stages  of  the  Plasmodium 
malarise  in  the  mosquito,  however,  are  well  known,  while  nothing  is 
known  of  such  stages  of  piroplasma  in  cattle  ticks.  Mosquitoes  do  not 
transmit  the  malarial  parasites  through  their  ova  to  their  offspring, 
but  the  eggs  of  a  tick  that  has  fed  upon  infected  cattle  will  develop 
ticks  that  spread  the  disease. 

A  knowledge  of  the  cattle  tick,  its  life  history  and  habits,  is  necessary 
in  the  campaign  to  limit  and  exterminate  the  disease,  and  for  this 
reason  the  description  given  by  Graybill  in  Farmer's  Bulletin  No. 
378,  United  States  Department  of  Agriculture,  Washington  Govern- 
ment Printing  Office,  1909,  is  here  inserted : 

"In  tracing  the  life  history  of  the  cattle  tick  it  will  be  convenient 
to  begin  with  a  large,  plump,  olive-green  female  tick,  somewhat 
more  than  half  an  inch  in  length,  attached  to  the  skin  of  the  host. 
During  the  few  preceding  days  she  has  increased  enormously  in 
size  as  a  consequence  of  drawing  a  large  supply  of  blood. 

"When  fully  engorged  she  drops  to  the  ground,  and  at  once,  espe- 
cially if  the  weather  is  warm,  begins  to  search  for  a  hiding  place  on 
moist  earth  beneath  leaves  or  any  other  litter  which  may  serve  as  a 
protection  from  the  sun  and  numerous  enemies.  The  female  tick 
may  be  devoured  by  birds  or  destroyed  by  ants,  or  may  perish  as  the 
result  of  unfavorable  conditions,  such  as  low  temperature,  absence 
or  excess  of  moisture,  and  many  other  conditions;  so  that  many 
which  fall  to  the  ground  are  destroyed  before  they  lay  eggs. 

"Egg-laying  begins  during  the  spring,  summer,  and  fall  months, 

1  Blood  is  defibrinated  in  the  following  manner:  Allow  blood  drawn  under  aseptic  precau- 
tions to  run  into  a  sterile  vessel  containing  some  glass  beads  or  fragments  of  glass.  Shake  well 
for  some  time.  The  fibrin  collects  around  and  clings  to  the  glass  pearls,  etc.,  and  the  defi- 
brinated blood  may  then  be  poured  ofr  into  another  sterile  vessel. 


Fio.  206 


PIG.  207 


FIG.  208 


FIG.  209 


FIG.  210 


FIGS.  204  to  210. — Cattle  ticks  in  various  stages.  Fig.  204.  Full-grown  female  tick,  engorged 
and  ready  to  drop  to  ground  and  deposit  eggs.  (Magnified  3  times.)  Fig.  205.  Tick  laying  eggs. 
One  tick  may  lay  as  many  as  5000  eggs.  (Magnified  3  times.)  Fig.  206.  Larva?  or  seed  ticks 
after  emerging  from  eggs.  (Magnified  9  times.)  Fig.  207.  Young  ticks  before  (a)  and  after  (6) 
first  molt.  At  this  stage  the  ticks  have  attached  themselves  to  a  host  (cow,  steer,  etc.),  and 
"have  changed  from  a  brown  color  to  white.  It  will  be  noticed  that  the  tick  has  six  legs  before 
molting  and  eight  afterward.  (Magnified  9  times.)  Fig.  208.  Young  tick  nearly  ready  to 
undergo  the  second  molt.  The  tick  at  this  stage  is  known  as  a  nymph.  (Magnified  6  times.) 
Fig.  209.  Male  tick.  (Magnified  6  times.)  Fig.  210.  Female  tick  after  second  molt.  This  tick 
is  now  sexually  mature  and  slightly  larger  than  the  male,  but  will  later  greatly  increase  in 
size  until  ready  to  drop  to  the  ground  and  deposit  eggs.  (Magnified  6  times.)  (Graybill.) 


588  PIROPLASMA  BOVIS 

in  from  two  to  twenty  days,  and  during  the  winter  months  in  thirteen 
to  ninety-eight  days.  The  eggs  are  small,  elliptical-shaped  bodies, 
at  first  of  a  light  amber  color,  later  changing  to  a  dark  brown,  and 
are  about  one-fiftieth  of  an  inch  in  length.  As  the  eggs  are  laid 
they  are  coated  with  a  sticky  secretion  which  causes  them  to  adhere 
in  clusters,  and  no  doubt  serves  the  purpose  of  keeping  them  from 
drying  out.  During  egg-laying  the  mother  tick  gradually  shrinks 
in  size  and  finally  is  reduced  to  about  one-third  or  one-fourth  of 
her  original  size.  Egg-laying  is  greatly  influenced  by  temperature, 
being  retarded  or  even  arrested  by  low  temperatures.  It  is  complete 
in  from  four  days  in  the  summer  to  one  hundred  and  fifty-one  days 
during  the  fall  and  the  beginning  of  winter.  During  this  time  the 
tick  may  deposit  from  a  few  hundred  to  more  than  5000  eggs. 
After  egg-laying  is  completed  the  mother  tick  has  fulfilled  her  purpose 
and  dies  in  the  course  of  a  few  days. 

"After  a  time,  ranging  from  nineteen  days  in  the  summer  to  one 
hundred  and  eighty-eight  days  during  the  fall  and  winter,  the  eggs 
begin  to  hatch.  From  each  egg  issues  a  small,  oval,  six-legged  larva, 
or  seed  tick,  at  first  amber  colored,  later  changing  to  a  rich  brown. 
The  seed  tick,  after  crawling  slowly  over  and  about  the  shell  from 
which  it  has  emerged,  usually  remains  more  or  less  quiescent  for 
several  days,  after  which  it  shows  great  activity,  especially  if  the 
weather  is  warm,  and  ascends  the  nearest  vegetation,  such  as  grass, 
herbs,  and  even  shrubs. 

"Since  each  female  lays  an  enormous  mass  of  eggs  at  one  spot, 
thousands  of  larvae  will  appear  in  the  course  of  time  at  the  same 
place  and  will  ascend  the  near-by  vegetation  and  collect  on  the 
leaves.  This  instinct  of  the  seed  tick  to  climb  upward  is  a  very 
important  adaptation  to  increase  their  chances  of  reaching  a  host. 
If  the  vegetation  upon  which  they  rest  is  disturbed  they  become 
very  active  and  extend  their  long  front  legs  upward  in  a  divergent 
position,  waving  them  violently  in  an  attempt  to  seize  hold  of  a  host. 

"The  seed  tick,  during  its  life  in  the  pasture,  takes  no  food,  and 
consequently  does  not  increase  in  size,  and  unless  it  reaches  a  host 
to  take  up  the  parasitic  portion  of  its  development  it  dies  of  star- 
vation. The  endurance  of  seed  ticks  is  very  great,  however,  as  they 
have  been  found  to  live  nearly  eight  months  during  the  colder  part  of 
the  year. 

"The  parasitic  phase  of  development  begins  when  the  larvae  or 
seed  ticks  reach  a  favorable  host,  such  as  a  cow.  They  crawl  up 
over  the  hair  of  the  host  and  commonly  attach  themselves  to  the  skin 
of  the  escutcheon,  the  inside  of  the  thighs  and  flanks,  and  to  the 
dewlap.  They  at  once  begin  to  draw  blood  and  soon  increase  in  size. 
In  a  few  days  the  young  tick  changes  from  a  brown  color  to  white, 
and  after  from  five  to  twelve  days  sheds  its  skin.  The  new  form  has 
eight  legs  instead  of  six,  and  is  known  as  a  nymph. 

"In  from  five  to  eleven  days  after  the   first   molt   the   tick  again 


EPIDEMIOLOGY  589 

sheds  its  skin  and  becomes  sexually  mature.  It  is  at  this  age  that 
males  and  females  are  with  certainty  distinguishable  for  the  first 
time.  The  males  emerge  from  the  skin  as  brown,  oval  ticks,  about 
one-tenth  of  an  inch  in  length.  He  has  reached  the  limit  of  growth 
and  goes  through  no  further  development.  Later  he  shows  great 
activity  in  moving  about  over  the  skin  of  the  host.  The  female  at 
the  time  of  molting  is  slightly  larger  than  the  male.  She  seldom 
shows  much  activity,  seldom  moving  far  from  her  original  point  of 
attachment.  She  still  has  to  undergo  most  of  her  growth.  After 
mating  the  female  increases  very  rapidly  in  size,  and  in  from  twenty- 
one  to  twenty-six  days  after  attaching  to  a  host  as  a  seed  tick  she 
becomes  fully  engorged  and  drops  to  the  ground  of  the  pasture,  to 
repeat  the  cycle  of  development. 

"To  sum  up,  on  the  pasture  there  are  found  three  stages  of  the 
tick — the  engorged  female,  the  egg,  and  the  larva;  and  on  the  host 
(cattle)  are  found  four  stages — the  larva,  the  nymph,  the  sexually 
mature  adult  of  both  sexes,  and  the  engorged  condition  of  the  female. 

"In  undertaking  measures  for  eradicating  the  tick  it  is  evident 
that  the  pest  may  be  attacked  in  two  locations,  namely,  on  the  pasture 
and  on  the  cattle. 

"In  freeing  pastures  the  method  followed  may  be  either  a  direct 
or  an  indirect  one.  The  former  consists  in  excluding  all  cattle, 
horses,  and  mules  from  pastures  until  all  the  ticks  have  died  of  star- 
vation. The  latter  consists  in  permitting  the  cattle  and  other  animals 
to  continue  on  the  infested  pasture  and  treating  them  at  regular 
intervals  with  oils  or  other  agents  destructive  to  ticks  and  thus  pre- 
venting engorged  females  from  dropping  and  reinfesting  the  pasture. 
The  larvae  on  the  pasture,  or  those  which  hatch  from  eggs  laid  by 
females  already  there,  will  all  eventually  meet  death.  Such  of  these 
as  get  upon  the  cattle  from  time  to  time  will  be  destroyed  by  the 
treatment,  while  those  which  fail  to  find  a  host  will  die  in  the  pasture 
from  starvation. 

"Animals  may  be  freed  of  ticks  in  two  ways.  They  may  be  treated 
by  solutions,  etc,,  that  will  destroy  all  the  ticks  present,  or  they  may 
be  rotated  at  proper  intervals  on  tick-free  fields  until  all  the  ticks 
have  dropped." 

Epidemiology. — A  number  of  points  in  the  epidemiology  of  Texas 
fever,  formerly  quite  mysterious  and  unexplainable,  are  now  easily 
understood,  since  the  etiology  of  the  disease  has  been  cleared  up. 
Wherever  Texas  fever  or  piroplasmosis  of  cattle  has  occurred  it 
was  observed  that  animals  on  the  pastures  are  more  commonly 
attacked  than  animals  kept  in  barns.  It  was  also  noticed  long  ago 
that  a  wet,  marshy  ground  upon  which  cattle  entered  in  spring 
formed  a  favorable  soil  for  the  appearance  of  the  disease.  Hot 
weather  favors  outbreaks  more  than  a  cool  temperature.  Animals 
born  and  raised  in  infected  territories  are  much  more  resistant  than 
animals  born  and  raised  in  a  free  territory  and  later  brought  to  the 


590  PIROPLASMA  BOVIS 

infected  territory.  This  is  due  to  the  fact  that  very  young  animals 
are  not  very  susceptible  to  the  disease;  they  acquire  it  in  a  mild 
form  and  a  certain  degree  of  immunity  becomes  established  by 
repeated  annual  infections.  The  immunity  so  acquired,  however,  is 
no  real  immunity,  but  simply  consists  in  the  presence  of  a  small 
number  of  piroplasmata  and  a  tolerance  against  them.  If  such 
animals  are  exposed  to  fatigue  (in  transit)  or  to  other  diseases  they 
may  develop  a  malignant  outbreak  of  the  disease. 

Such  partially  immune  animals  if  brought  to  a  non-infected 
district  may  become  the  source  of  violent  outbreaks  among  the 
animals  of  the  hitherto  free  territory.  Transmission  in  these  cases 
may  be  brought  about  by  either  one  of  two  ways:  (1)  The  animals 
from  the  infected  district  may  carry  with  them  infected  ticks  which 
will  directly  spread  the  disease  in  a  hitherto  free  territory,  or  (2)  they 
may  not  bring  ticks  along,  but  find  in  their  new  surroundings  ticks 
which  can  and  will  spread  the  disease. 

Immunization  of  Cattle. — It  was  noticed  by  Smith  and  Kilbourne 
that  animals  after  recovery  from  an  attack  of  Texas  fever  in  one 
year  were  comparatively  immune  against  new  attacks  in  subsequent 
years  in  spite  of  being  much  exposed  to  infected  ticks.  Schroeder 
was  one  of  the  first  in  this  country  experimentally  to  inoculate  young 
northern  cattle  with  blood  from  infected  Southern  animals,  producing 
by  this  method  a  mild  attack  of  Texas  fever.  Subsequently  he 
exposed  the  inoculated  animals  together  with  non-protected  control 
animals  in  the  South  to  the  natural  tick  infection.  The  results  were 
very  favorable  and  promising :  most  of  the  protected  animals  lived, 
and  all  the  controls  died. 

Dalrymple1  has  published  a  report  on  the  results  and  experiences 
of  protective  inoculation  of  cattle  against  Texas  fever.  He  states 
(1)  that  sterile  blood  serum  of  infected  animals  obtained  by  centri- 
fuging  the  blood  and  separating  the  corpuscles  from  the  serum, 
has  no  value  whatever  in  immunizing  animals;  (2)  that  susceptible 
cattle  may  be  immunized  by  infecting  them  with  piroplasma  through 
the  medium  of  infected  seed  ticks,  but  on  account  of  certain  trouble- 
some conditions  the  method  is  not  as  practical  as  could  be  desired. 
The  results  of  experiments  to  utilize  the  blood  from  ticks  in  immuni- 
zing inoculations  is  summarized  as  follows  by  Dalrymple: 

"The  blood  with  which  the  adult  ticks  are  filled,  after  maturing 
on  Southern  cattle,  carries  with  it  the  power  to  produce  Texas  fever 
when  injected  under  the  skin  of  a  susceptible  animal. 

"Experiments  indicate  that  we  may  be  able  to  take  ticks  from 
recently  immunized  animals,  ship  them  considerable  distances,  and 
utilize  them  as  a  substitute  for  the  blood  drawn  from  the  vein,  where 
recently  immunized  animals  are  not  available. 

"Experiments  further  indicate  that   this  will  give  a  milder  form 

1  Louisiana  State  University  and  A.  and  N.  College  Bulletin,  No.  84,  October,  1905. 


IMMUNIZATION  OF  CATTLE  591 

of  the  disease,  and  afterward,  immunty  just  as  effectual  as  when 
the  blood  is  taken  from  an  immune  animal  immediately  before  being 
used. 

"We  have  not  as  yet  found  any  way  of  preserving  the  blood  drawn 
from  the  vein  for  any  considerable  time  without  its  losing  its  power 
to  produce  immunity." 

The  Louisiana  report  of  Dalrymple  gives  the  following  conclusions 
and  directions  as  to  the  immunizing  of  Northern  cattle  by  the  use 
of  fresh  blood  from  infected  animals: 

"Previous  to  the  discovery  and  adoption  of  the  blood-inoculation 
method  of  immunizing  susceptible  Northern  cattle  against  the  ravages 
of  Texas  fever  the  mortality  in  these  animals  ranged  anywhere 
from  40  to  90  per  cent.  This,  necessarily,  discouraged  Southern 
stockmen  in  the  importation  of  pure-bred  cattle  for  the  purpose  of 
improving  their  herds,  and  accounts,  mainly,  for  the  scarcity  of 
pure  and  highbred  stock  in  the  South  up  to  within  recent  years. 

"Consequent  upon  the  use  of  this  artificial  method  of  immuni- 
zation, however,  the  death  rate  from  the  fever  has  been  enormously 
reduced.  In  a  bulletin  issued  by  the  Texas  Experiment  Station  in 
1902  a  record  was  compiled  showing  the  percentage  mortality  of 
inoculated  cattle  that  had  been  treated  at  the  Texas,  Louisiana,  and 
other  Southern  (including  Missouri)  stations,  which  comprised  several 
thousand  head  (4562  up  to  January  1,  1904),  to  be  only  7.7,  and 
that,  too,  under  various  conditions  of  treatment  after  they  had 
been  placed  in  their  owner's  care.  This  record  has  given  increased 
encouragement  to  cattle  men  in  the  South. 

"The  technique  of  the  operation  as  practised  in  the  Louisiana 
Experiment  Station  is  the  following:  The  supply  animal  from 
which  the  immunizing  infected  blood  is  used  is  either  a  native  or 
a  Northern  immune  which  should  be  in  robust  health  and  condition. 
Experiments  and  experience  seem  to  indicate  that  the  most  suitable 
subjects  for  immunization  are  cattle  from  eight  to  twelve  months  old, 
in  good  flesh,  and  weight  from  500  to  800  pounds.  Before  inoculation 
it  is  well  to  allow  the  animal  to  rest  for  a  few  days,  especially  those 
that  may  have  come  off  a  tedious  railroad  journey;  and  during  this 
time  they  should  be  well  and  carefully  fed  and  kept  absolutely  free 
from  ticks. 

"The  operation  seems  more  easily  performed  with  the  supply 
animal  thrown  down  and  tied.  The  hair  is  clipped  from  a  portion 
of  the  skin  of  the  neck  just  over  the  jugular  vein.  The  denuded  part 
is  bathed  with  an  antiseptic  solution.  The  neck  of  the  animal  is 
now  straightened  and  tensed,  and  a  piece  of  strong  cord  or  small 
rope  tied  around  its  base  sufficiently  tight  to  check  the  flow  of  blood 
and  raise  the  vein.  A  small  trocar  and  cannula,  or  hollow  sharp- 
pointed  needle,  which  has  previously  been  sterilized,  or  disinfected, 
is  then  inserted  into  the  distended  vein  and  is  directed  up  the  vessel 
toward  the  head.  As  soon  as  the  needle  enters  the  vein  the  blood 


592  PIROPLASMA  BOVIS 

passes  through  it  into  a  sterilized  glass,  and  when  sufficient  blood 
has  been  obtained  the  needle  or  cannula  is  withdrawn,  the  rope 
around  the  neck  loosened,  and  the  wound  bathed  with  antiseptic 
solution. 

"To  prevent  the  blood  from  clotting,  after  it  has  been  withdrawn 
it  is  immediately  stirred  slowly  with  a  thin  glass  rod,  which  has 
been  disinfected,  until  as  much  as  possible  of  the  clot  has  collected 
on  it,  and  it  is  then  withdrawn.  The  remaining  defibrinating  blood, 
or  a  part  of  it,  is  then  drawn  up  into  a  clean  hypodermic  syringe, 
and  the  quantity  to  be  injected  gauged  by  a  small  screw-regulator 
on  the  piston. 

"The  animal  to  be  inoculated  is  prepared  by  clipping  the  hair 
from  off  a  portion  of  the  skin — about  the  size  of  the  hand — behind 
or  a  little  above  the  point  of  the  elbow,  on  the  side  of  the  chest; 
any  part  where  the  skin  is  loose  and  thin  will  answer.  This  is  dis- 
infected as  in  the  case  of  the  supply  animal.  The  skin  is  then  drawn 
out  between  the  thumb  and  forefinger  of  the  left  hand  and  an  incision 
made  through  it  with  a  narrow,  sharp-pointed  knife  or  lancet,  to 
allow  of  easy  introduction  of  the  hypodermic  needle,  care  being  taken 
not  to  injure  the  chest  wall.  If  the  needle  is  a  strong  one  it  may  not 
be  necessary  to  use  the  lance.  The  syringe  is  now  attached  to  the 
needle  and  the  required  amount  of  blood  injected  under  the  skin. 
After  withdrawal  of  the  needle  the  part  is  again  lightly  bathed 
with  the  antiseptic  solution.  Success  depends  very  largely  upon  the 
antiseptic  precautions  taken  in  the  operation.  Consequently,  all 
instruments  and  utensils,  the  hands  of  the  operator  and  the  operative 
area  of  skin  should  be  thoroughly  disinfected. 

"The  standard  amount  of  blood  used  at  the  Louisiana  Station  for 
some  time  has  been  1  cubic  centimenter  (about  16  drops)  for  animals 
of  any  age.  Latterly,  however,  it  has  been  customary  to  administer 
a  second  dose  of  2  cubic  centimeters  after  recovery  from  the  fever 
period.  The  object  is  to  increase  if  possible  the  degree  of  immunity 
before  the  animal  is  exposed  to  ticks.  After  the  second  injection 
of  blood  the  patient  is  kept  under  observation  for  ten  days  or  some- 
what longer,  and  the  temperature  taken  to  watch  the  course  of  the 
fever,  should  there  be  any. 

"We  have  previously  stated  that  the  blood  before  injection  was 
stirred  to  remove  the  clot  (defibrinated).  This  is  usually  done 
when  a  number  of  animals  are  to  be  inoculated,  to  prevent  clotting 
before  the  work  is  completed.  In  the  case  of  a  single  animal,  how- 
ever, or  even  two  or  three,  the  blood  may  be  drawn  directly  from 
the  vein  of  the  supply  animal  into  the  hypodermic  syringe  and  injected 
immediately  into  the  other  animal  or  animals/' 

Although  inoculation  may  be  performed  at  any  season  of  the 
year,  the  best  time  in  the  Southern  climate  is  during  the  late  fall  or 
early  winter  months.  This  prevents  a  too  sudden  gross  infection 
with  ticks  when  the  animal  is  turned  into  pasture,  as  would  naturally 
be  the  case  during  the  summer  months. 


PIROPLASMA  CANIS  593 

Kaumanns,  in  a  paper  read  before  the  1909  (Chicago)  Meeting 
of  the  American  Veterinary  Association,  and  published  in  full  in  the 
Proceedings  of  the  Association,  strongly  advocates  the  crossing  of 
American  cattle  with  Brahma  cattle  from  India  for  the  purpose  of 
producing  a  natural  immunity.  He  states  that  only  35  per  cent, 
of  Indian  blood  is  required  to  produce  a  race  almost  entirely  immune 
against  Piroplasma  bigeminum  infection.  The  great  difficulty  in 
carrying  out  such  a  plan  consists  in  getting  Indian  cattle  free  from 
latent  trypanosoma  infection.  In  attempts  made  in  the  past  it  was 
found  that  a  high  perentage  of  such  cattle,  though  examined  thor- 
oughly before  exportation  from  India,  were  found  infected  with  surra 
upon  arrival  in  the  United  States.  Evidently  the  journey  and  the 
change  of  climate  awakened  the  latent  trypanosomiasis. 


PIROPLASMA  CANIS. 

The  piroplasma  infection  in  dogs  was  first  described  by  Piana  and 
Galli-Valerio  in  Italy,  in  1895.  The  two  investigators  recognized 
the  similarity  between  the  parasites  in  the  dog's  blood  corpuscles 
and  those  in  Texas  fever,  and  called  them  Piroplasma  bigeminum, 
var.  canis.  They  were  subsequently  found  in  dogs  by  a  number  of 
authors  and  their  morphology  has  been  studied  particularly  by 
Schilling,  Nuttal  and  Graham,  Kinoshita,  Bowhill,  LeDuc,  Chris- 
topher, and  others.  In  several  respects  they  are  the  best-known 
representatives  of  the  family  piroplasma  or  babesia. 

In  the  fresh  blood  of  the  dog  the  intracorpuscular  parasite  can 
best  be  seen  at  the  height  of  the  fever;  it  appears  in  the  inside  of 
the  erythrocyte  as  a  light,  highly  refractive,  sharply  defined  body, 
generally  found  in  somewhat  enlarged  red  blood  corpuscles.  If 
examined  in  fresh  blood  on  a  warm  stage  the  protoplasm  of  the 
parasite  shows  some  contractility  and  appears  to  send  out  fine  pro- 
cesses. The  latter  may  be  so  fine  that  they  look  like  flagella.  The 
largest  forms  of  the  parasites  are  found  in  freshly  infected  dogs,  the 
smallest  in  old  chronic  cases.  Blood  smears  stained  with  Roman- 
owski's,  Wright's,  or  Giemsa's  stain  show  a  small  amount  of  oval 
or  pear-shaped,  round  or  ring-shaped  protoplasm  stained  bluish 
with  a  dot  stained  red.  The  latter  is  the  nucleus  of  the  parasite. 
The  nucleus  is  not  always  small,  sometimes  it  is  quite  large,  and 
occupies  a  considerable  portion  of  the  cytoplasm  of  the  piroplasm. 
These  forms,  after  the  fever  has  reached  its  height,  and  when  the 
temperature  is  going  down,  are  also  found  outside  of  red  blood  cor- 
puscles in  the  blood  plasma.  The  microorganism  is  also  found  in 
the  internal  organs,  particularly  in  the  liver,  the  kidneys,  and  the 
bone  marrow. 

The  nucleus  of  Piroplasma  canis  is  of  indefinite  shape,  composed 
of  chromatin  granules  varying  in  form;  it  is  generally  situated  eccen- 
38 


594 


PIROPLASMA  CANIS 


trically  in  the  intracorpuscular  forms,  but  in  the  centre  of  the  para- 
sites which  are  found  free  in  the  blood  plasma. 

The  Piroplasma  canis,  like  the  bigeminum,  reproduces  in  the 
interior  of  red  blood  corpuscles.  According  to  Nuttall  and  Smith 
a  small,  round,  young  form  is  first  present.  This  becomes  larger, 
begins  to  divide  by  becoming  somewhat  saddle-shaped  and  pear- 
shaped,  with  the  twin  arrangement  later  on.  The  twins  after  being 
completely  divided  leave  the  red  blood  corpuscles,  enter  new  ones, 
and  repeat  the  cycle. 

Kinoshita  has  described  an  irregular  division  or  budding  process 
which  he  regards  as  an  asexual  multiple  reproduction  (schizogony), 
with  merozoite  formation,  as  it  occurs  in  malarial  organisms.  Chris- 
topher, however,  claims  that  such  a  mode  of  division  does  not  occur 
in  piroplasmata,  that  they  divide  always  into  two  equal  halves,  but 
that  subdivision  may  start  before  the  first  division  is  complete. 


FIG.  211 


Stages  in  the  development  of  Babesia  canis.  (After  Kinoshita.)  At  round  discoid  parasite 
in  a  blood  corpuscle;  B,  ameboid  form,  with  long  processes;  C,  a  pair  of  "mature"  gametes; 
D,  a  mature  "female"  gamete;  E,  a  mature  "male"  gamete;  F.  a  budding  form  in  blood  cor- 
puscles; G,  a  group  of  sixteen  "young"  gametes. 

The  most  constant  symptom  in  canine  piroplasmosis  is  hematuria, 
just  as  in  Texas  fever  of  cattle. 

Natural  infection  is  brought  about  by  biting  ticks.  Several  kinds 
have  been  named  as  being  the  intermediate  host  of  Piroplasma  canis, 
among  them  Hemophalis  leachii,  which,  however,  can  only  transmit 
the  disease  during  the  mature  stage;  neither  the  six-legged  larvae 
nor  the  eight-legged  nymphse  can  transfer  the  disease  from  infected 
to  healthy  dogs. 


PIROPLASMOSIS  OF  HORSES  595 


PIROPLASMA  OVIS. 

Sheep  are  subject  to  a  disease  known  as  icterohematuria,  or  hemo- 
globinuria.  It  is,  like  the  preceding  affections  of  cattle  and  dogs,  a 
piroplasmosis.  The  intracorpuscular  organisms  were  first  seen  by 
Babes  in  1888,  who,  however,  did  not  recognize  their  protozoan 
nature.  Piroplasmosis  in  sheep  was  subsequently  described  by 
Bonome  in  Italy,  Nicolle  in  Turkey,  Leblanc  in  France,  and  Hutche- 
son  in  Transvaal.  The  most  marked  pathologic  changes  in  piro- 
plasmosis in  sheep  are  a  serous  infiltration  of  the  subcutaneous, 
retroperitoneal,  and  mediastinal  connective  tissue,  inflammatory  and 
hemorrhagic  changes  in  the  gastro-intestinal  tract,  enlargement 
and  great  congestion  of  the  spleen,  parenchymatous  degeneration  and 
necrosis  of  the  liver,  parenchymatous  degeneration  of  the  kidneys, 
and  multiple  subserous  and  submucous  hemorrhages.  The  bladder 
contains  a  bloody  urine.  The  parasites  causing  the  disease  are 
piroplasmata  of  the  usual  shape  and  structure.  Dividing  forms  can 
best  be  seen  in  juice  expressed  from  the  spleen.  The  anemia  caused 
during  the  short  course  of  the  disease  is  very  profound,  and  the  count 
of  erythrocytes  sinks  from  8,000,000  to  1,500,000.  The  mortality  of 
the  disease  is  very  high;  recovered  animals  are  said  to  be  immune. 
The  disease  is  spread  by  ticks. 


PIROPLASMOSIS  OF  HORSES. 

Besides  the  infectious  anemia  in  horses,  which  is  a  disease  evidently 
due  to  an  ultramicroscopic  filterable  organism  concerning  which 
absolutely  nothing  is  known,  anemias  in  horses  due  to  piroplasma 
infection  have  been  observed  at  various  times  and  places.  Ziemann 
claims  to  have  seen  intracorpuscular  piroplasmata  in  the  blood  of 
horses  sick  with  hematuria  in  Germany.  Hutcheson  reported  similar 
findings  from  Cape  Colony.  Bordet  and  Danysz  found  equine  piro- 
plasmosis in  Transvaal.  Some  authors  have  claimed  that  the  Plas- 
modium  malarias  has  also  been  found  in  horses,  but  this  is  denied 
by  Laveran,  who  holds  that  all  intracorpuscular  parasites  found  in 
the  blood  of  horses  are  piroplasma  and  none  Plasmodium  malarise. 
Some  of  the  later  observations  on  equine  piroplasmosis  were  made 
by  Pallin  in  India  and  Robert  Koch  and  Theiler  in  Africa.  The 
latter  distinguishes  two  types :  a  mild  form,  where  a  diagnosis  can  only 
be  made  by  finding  a  few  piroplasmata  in  the  blood,  and  serious  cases, 
with  high  fever  and  marked  sickness.  The  former  type  generally 
ends  in  recovery,  the  latter  in  death.  The  parasites  are  found  in 
the  blood  and  most  abundantly  in  the  spleen.  The  organisms  are 
generally  round,  have  a  diameter  from  0.5  to  2.5,  and  look  a  good 
deal  like  tropical  and  tertiary  malarial  parasites.  Pear-shaped  forms 


596  PIROPLASMOSIS  OF  HORSES 

in  couples  are,  however,  also  seen.  Romanowski's  stain  shows  a 
red-stained  chromatin  granule  near  the  periphery  of  the  parasites; 
pigment  (as  in  malarial  parasites)  is  never  seen.  Laveran  described 
amitotic  division  of  the  nucleus;  two  divisions  may  follow  each  other 
rapidly,  so  that  four  parasites  arranged  in  rosette  form  may  be  seen 
in  a  red  blood  corpuscle.  The  most  prominent  changes  in  equine 
piroplasmosis  are  intense  jaundice,  with  enormous  enlargement  of 
the  spleen,  which  may  weigh  ten  pounds  or  more.  The  pulp  of  the 
spleen  is  very  soft,  dark,  and  tar-like.  The  bladder  contains  hemor- 
rhagic  urine.  Subserous  and  submucous  hemorrhages  are  frequently 
seen;  the  heart  muscle  is  very  soft  and  flabby,  and  ruptures  easily. 
The  disease  is  spread,  like  the  other  piroplasmoses,  by  ticks. 


QUESTIONS 

1.  What  kind  of  a  disease  is  Texas  fever? 

2.  What  other  names  have  been  given  to  this  disease? 

3.  What  were  the  claims  of  Babes  as  to  the  nature  and  cause  of  the  disease? 

4.  What  is  the  real  cause  of  Texas  fever?    Who  discovered  it? 

5.  Describe  the  pathologic  changes  in  a  very  acute  case  of  Texas  fever. 

6.  What  are  the  blood  changes  in  Texas  fever? 

7.  What  is  the  meaning  of  the  terms  oligocythemia  and  oligochromemia  ? 

8.  How  can  an  absolute  diagnosis  of  Texas  fever  be  made? 

9.  Describe  the  steps  in  a  blood  examination  for  establishing  a  diagnosis  of 
Texas  fever. 

10.  Describe  the  morphology  of  the  Piroplasma  bigeminum. 

11.  What  is  the  relation  between  the  parasites  and  febrile  temperatures  in 
infected  animals? 

12.  Where  are  the  piroplasmata  found  most  numerous  after  the  death  of  an 
infected  animal? 

13.  How  long  does  blood  infected  with  piroplasmata  remain  virulent  under 
various  conditions? 

14.  Describe  the  cultural  properties  of  Piroplasma  bigeminum  and  the  prepar- 
ation of  the  proper  culture  medium. 

Li  15.  What  is  the  natural  mode  of  transmission  in  Texas  fever? 
""*  16.  What  animals  are  susceptible  to  this  disease? 
3f  17.  How  can  the  disease  be  transferred  artificially? 

'  18.  What  are  the  technical  names  of  the  Texas  fever  cattle  tick?    Describe 
its  life  history. 

19.  What  methods  have  been  practised  to  immunize  cattle  against  Texas 
fever? 

20.  Describe  in  detail  the  Louisiana  method. 

21.  When  is  the  best  time  to  immunize  animals  against  Texas  fever? 

22.  What  animal  is  the  host  of  Piroplasma  canis?    Describe  the  morphology 
of  the  latter. 

23.  Describe  its  method  of  reproduction. 

24.  Discuss  the  different  views  as  to  processes  of  reproduction  in  Piroplasma 
canis. 

25.  What  is  the  most  constant  symptom  in  piroplasmosis  of  dogs? 

26.  How  is  the  disease  spread? 

27.  What  difference  in  the  mode  of  spreading  is  there  between  piroplasmosis 
of  dogs  and  Texas  fever? 

28.  Describe  the  most  prominent  pathologic  changes  of  ictero  hematuria  in 
sheep.     What  is  the  cause  of  the  disease  ? 

29.  What  are  the  most  prominent  pathologic  changes  in  piroplasmosis  of 
horses?     Where  has  the  disease  been  observed? 


CHAPTEE    LV. 

RABIES  AND  THE  NEGRI  BODIES  (NEURORYCTES  HYDROPHOBIA). 

RABIES,  lyssa,  hydrophobia,  canine  madness,  "Wasserscheu," 
"Tollwuth,"  "Hundswuth"  (German),  "rage"  (French),  is  an  acute 
contagious,  generally  fatal  disease  of  wolves,  foxes,  dogs,  and  more 
rarely  of  other  domestic  animals  and  man.  It  is  due  to  a  specific 
virus,  which,  with  the  infective  saliva,  gains  entrance  into  the  body  of 
a  susceptible  being  through  a  wound  generally  caused  by  the  bite 
of  some  animal  suffering  from  the  disease. 

Historical  and  Occurrence. — Rabies  among  dogs  and  the  danger  to 
other  animals  from  the  bite  of  a  rabid  dog  were  known  to  Aristoteles, 
the  Greek  naturalist  and  philosopher.  That  the  saliva  of  such 
animals  was  the  carrier  of  the  infective  agent  was  shown  experiment- 
ally by  Zinke,  Gruner,  and  Salm  in  the  early  part  of  the  last  century. 
Galtier,  in  1879,  was  the  first  to  inoculate  rabbits,  and  in  1881  Pasteur 
and  his  co-workers,  Roux,  Chamberland,  and  Thuilliers,  became  the 
main  exponents  in  the  modern  study  of  hydrophobia  upon  which  is 
based  our  exact  knowledge  of  the  disease  and  the  methods  of  its 
prevention  by  the  inoculation  of  an  attenuated  virus.  The  disease  has 
been  encountered  almost  all  over  the  world,  but  has  apparently  been 
kept  out  of  Australia.  It  has  been  on  the  increase  during  the  last 
decade  or  two  in  the  United  States. 

Natural  Infection. — Natural  infection,  as  a  rule,  is  caused  by  the 
bite  of  animals  suffering  from  the  disease,  but  sometimes  dogs  in  the 
early  stages  of  unrecognized  rabies  have  inoculated  persons  by  licking 
a  place  where  there  is  a  small  abrasion  of  the  skin.  The  saliva  is 
most  infective  after  the  disease  has  well  developed  and  during  its 
subsequent  course,  but  may  also  be  infective  before  any  symptoms 
of  rabies  appear.  The  danger  of  the  bite  from  a  rabid  animal  depends 
upon  the  greater  or  lesser  virulency  of  the  saliva,  upon  the  extent  of  the 
wound,  the  amount  of  laceration,  the  vascularity  and  nerve  supply 
of  the  tissue,  and  upon  the  greater  or  lesser  distance  of  the  wound 
from  the  central  nervous  system.  The  less  the  distance  the  greater 
the  danger;  hence,  wounds  of  the  face  or  head  are  particularly  dan- 
gerous. Horses  and  cattle  are  especially  liable  to  contract  hydro- 
phobia if  bitten  by  rabid  dogs,  wolves,  or  foxes  in  the  lips,  cheeks,  or 
nose.  The  danger  of  the  bite  is  much  lessened  if  the  parts  are  covered 
by  a  dense  fur,  or,  in  the  case  of  man,  by  heavy  clothing.  It  has 
been  shown  that  shorn  sheep  are  much  more  liable  to  develop  rabies 
after  being  bitten  than  those  covered  with  a  dense  wool.  The  virus, 


598  RABIES  AND  THE  NEGRI  BODIES 

so  far  as  known,  cannot  penetrate  the  intact  skin,  but  rabbits  may 
be  infected  through  the  intact  nasal  and  conjunctival  mucosa.  The 
virus  cannot  enter  through  the  gastro-intestinal  tract,  as  has  been 
shown  by  Nocard  in  his  feeding  experiments.  The  percentage  of 
infections  in  animals  bitten  by  dogs  suffering  from  rabies  is  variously 
estimated,  and  the  figures  given  extend  over  a  wide  range,  anywhere 
from  5  to  40  per  cent.  There  appears  to  be  both  a  racial  and  an 
individual  variability.  The  virus  of  hydrophobia  after  having  gained 
access  to  the  body  of  a  susceptible  animal  travels  from  the  portal  of 
entrance  to  the  central  nervous  system,  where  it  spreads  gradually. 
According  to  the  investigations  of  Vestea  and  Zagari,  inoculation 
into  the  sciatic  nerve  of  the  rabbit  is  first  followed  by  paralysis  of  the 
hind  leg  of  the  same  side  and  the  paralysis  progresses  from  behind 
to  the  anterior  part  of  the  body.  When  the  inoculation  is  made  in 
an  anterior  extremity  the  progress  is  from  in  front  backward.  The 
virus  multiplying  in  the  central  nervous  system  affects  the  vessel 
walls  and  produces  around  them  small,  round-celled  inflammatory 
infiltrations.  It  damages  the  ganglion  cells,  causing  psychical  dis- 
turbances, increase  of  reflex  irritability,  and  later  degenerative 
manifestations,  with  paralysis,  which  finally  affects  the  respiratory 
apparatus  and  so  leads  to  death. 

Period  of  Incubation. — One  of  the  most  remarkable  characteristics 
of  hydrophobia  is  the  great  variability  of  the  period  of  incubation 
after  natural  infection.  As  a  rule,  this  period  lasts  a  few  weeks,  but 
not  infrequently  may  be  prolonged  to  a  few  months.  In  dogs  and 
hogs  the  period  of  incubation  is  frequently  shortened  to  ten  to  fifteen 
days;  in  horses  and  cattle  it  is  frequently  from  one  to  three  months. 
According  to  statistics  by  Roell,  of  144  rabid  dogs,  43  per  cent, 
developed  manifest  symptoms  of  hydrophobia  within  thirty  days 
after  being  bitten,  40  per  cent,  between  the  thirtieth  to  the  sixtieth 
day,  14  per  cent,  between  the  sixtieth  to  the  nintieth  day,  and  3  per 
cent,  between  the  fourth  to  twelfth  month.  Zuendel  gives  the  following 
figures  for  579  head  of  cattle:  5  per  cent,  in  less  than  fifteen  days, 
23  per  cent,  between  fifteen  and  thirty  days,  39  per  cent,  between 
thirty  to  forty-five  days,  13  per  cent,  between  forty-five  to  sixty  days, 
17  per  cent,  between  three  to  six  months.  One  animal  after  forty-two 
and  one  after  ninety-five  weeks.  Unusually  long  periods  of  incubation 
in  horses  have  been  reported;  by  Gosswinter,  twenty  months;  by 
Bahr,  twenty-one  months;  by  Swain,  twenty-five  months;  in  cattle 
by  Szabo,  three  hundred  and  twenty-three  days;  Mieckley,  three 
hundred  and  twenty-seven  days;  Leipert,  nearly  twenty  months;  Kalt, 
twenty-three  months.  Ligniere  reported  the  case  of  a  rabid  watch  dog 
on  a  farm  biting  twenty  head  of  cattle.  Four  oxen  out  of  the  twenty 
animals  bitten  died  after  incubation  periods  between  two  and  six 
months.  All  four  succumbed  to  the  paralytic  form  of  the  disease,  as 
also  did  one  cow,  but  only  after  an  incubation  period  lasting  three  years 
In  man  the  period  of  incubation  likewise  varies  considerably.  Paltauf , 


PERIOD  OF  INCUBATION  590 

who  has  recently  furnished  a  very  interesting  contribution  to  the  patho- 
genesis  of  rabies  in  man,  states  that  the  shortest  period  on  record  is 
about  fourteen  days;  the  average  period,  eight  to  twelve  weeks.  This 
author  has  seen  a  case  with  a  period  of  incubation  of  twenty  months; 
Spencer  one  of  two  years,  four  months,  and  Krikoff  one  of  three 
years  and  two  and  one-half  months.  While  all  fully  developed 
cases  of  rabies  in  man  lead  to  a  fatal  issue,  man  is  not  very  susceptible 
to  the  disease.  According  to  older  statistics  dealing  with  persons 
bitten  by  rabid  dogs,  16  to  20  per  cent,  develop  the  disease.  Hoegyes 
gives  a  percentage  figure  of  13.9,  other  authors  of  5  to  6  per  cent. 
Kirchner's  figure  for  Germany  is  2  per  cent,  to  3  per  cent.,  and 
Paltauf  thinks  that  the  figure  is  certainly  below  10  per  cent.  These 
percentages  all  refer  to  persons  bitten  by  rabid  dogs.  The  more 
serious  lacerations  produced  by  rabid  wolves  are  much  more  danger- 
ous, and  it  is  estimated  that  60  per  cent,  of  persons  bitten  by  these 
animals  contract  the  disease  and  die  from  it  if  not  treated. 

Paltauf's  recent  studies  of  the  pathology  and  pathogenesis  of 
rabies  in  man  were  made  on  four  persons  who  died  shortly  after  being 
bitten  by  rabid  dogs  from  some  intercurrent  diseases  which  had 
nothing  at  all  to  do  with  hydrophobia. 

In  all  four  cases  it  was  found  that  the  medulla,  when  emulsified 
and  injected  subdurally,  infected  rabbits  with  rabies,  showing  the 
presence  of  active  virus  in  the  patients'  nervous  tissues;  but  this 
virus  was  in  an  attenuated  condition,  since  the  incubation  period 
in  the  inoculated  rabbits  was  unusually  long,  forty  days  and  over, 
and  the  type  of  rabies  developed  was  that  of  the  chronic  or  "con- 
sumptive" form.  As  probably  at  the  highest  estimate  but  one  person 
in  ten  of  those  bitten  develops  rabies,  it  must  be  assumed  that  four 
consecutive  positive  findings  do  not  represent  latent  infection  which 
would  have  manifested  itself  had  the  patients  lived  longer.  It  appears 
rather  that  these  observations  indicate  that  in  persons  bitten  by 
rabid  animals  the  virus  commonly  reaches  the  central  nervous 
system,  but  that  nine  times  in  ten  it  is  there  destroyed  by  the  natural 
defensive  agencies  without  causing  symptoms.  These  agencies  may 
be  made  more  effective  by  the  immunizing  process  of  the  Pasteur 
treatment.  In  other  words,  rabies-inoculated  men  usually  develop 
a  latent  infection  which  is  overcome  without  the  symptoms  of  rabies. 
The  medulla  of  three  other  persons,  who  died  shortly  after  the  com- 
pletion of  a  course  of  Pasteur  treatment, were  found  to  be  non-infectious 
for  rabbits,  indicating  that  the  virus  was  destroyed  under  the  influence 
of  the  immunization. 

Presumably  the  virulency  of  the  virus  with  which  the  individual 
is  inoculated  is  one  of  the  main  factors  in  deciding  whether  it  will 
be  overcome  or  not,  for  the  bites  of  rabid  wolves  cause  rabies  in 
about  60  per  cent,  of  those  bitten  as  against  from  6  to  10  per  cent,  or 
less  of  fatalities  from  dog  bites,  and  none  from  subcutaneous  inocula- 
tion of  attenuated  rabbit  virus.  Possibly  in  other  cases  the  failure 


600  RABIES  AND  THE  NEGRI  BODIES 

to  destroy  the  virus  may  depend  on  individual  lack  of  immunizing 
power,  and  sometimes  on  local  disturbances  in  the  central  nervous 
system.  Medical  literature  is  full  of  records  of  cases  in  which  either 
physical  or  mental  shock  seemed  to  determine  the  development  of 
rabies. 

Paltauf,  from  his  studies  and  the  observations  given  above,  draws 
the  following  summary  of  conclusions  with  reference  to  the  behavior 
of  the  virus  of  rabies  after  its  entrance  in  natural  infection  into  the 
bodies  of  animals  and  persons : 

1.  The  period  of  incubation  in  rabies  in  man  varies  between  two 
weeks  and  two  years  and  over. 

2.  Dogs  are  evidently  quite  susceptible  to  the  virus  of  rabies, 
though  not  highly  so,  since  about  40  per  cent,  of  dogs  bitten  contract 
the  disease. 

3.  Man  is  not  very  susceptible  to  the  virus  of  rabies  in  dogs;  only 
from  6  to  9  per  cent,  or  perhaps  even  a  much  smaller  number  of 
persons  bitten  develop  rabies,  and  if  not  properly  treated  early,  die 
from  the  disease. 

4.  Of  persons  bitten  by  wolves  about  60  per  cent,  develop  rabies. 

5.  There   is   an  enormous   difference   between   the   original  sus- 
ceptibility of  man  to  the  virus  of  rabies  in  dogs  and  the  mortality 
in  cases  which  have  proved  to  be  susceptible. 

6.  Observations  made  on  four  persons  bitten  by  rabid  dogs  and 
who  died  from  intercurrent  diseases   (not  rabies)   soon  after   the 
Pasteur  treatment  was  begun  showed  that  their  cords  when  emulsified 
and  injected  into  rabbits  produced  after  long  periods  of  incubation 
the  slow,  so-called  "consumptive"  form  of  rabies. 

Paltauf  does  not  try  to  explain  the  long  period  of  incubation  of 
two  to  three  years  and  more  which  has  been  observed  in  a  few  authen- 
ticated cases  of  rabies  in  animals  and  persons.  It  might  be  explained, 
perhaps,  as  follows:  When  animals  or  persons  are  bitten  by  rabid 
dogs  there  may  be  a  shorter  or  longer  period  of  latency,  and  the 
virus  may  finally  be  completely  exterminated  by  the  protective  powers 
of  the  body.  On  the  other  hand,  there  may  occur  an  incomplete 
destruction  of  the  virus  and  imperfect  immunity,  but  a  continuous 
period  of  latency  as  is  found  in  trypanosomiasis  and  particularly  in 
Texas  fever.  In  the  latter  disease  a  few  piroplasmata  may  persist  in 
cattle  without  detriment  to  the  animal  until  a  great  strain,  changes 
in  environment,  or  intercurrent  disease  leads  to  a  sudden  multi- 
plication of  these  organisms,  with  a  typical  explosive  outbreak  of  the 
disease.  Likewise,  it  is  quite  possible  that  the  rabies  organisms 
may  survive  for  a  long  time  in  small  numbers  in  a  person  bitten  by 
a  mad  dog,  and  that  under  certain  conditions  they  may  suddenly 
multiply  and  produce  a  typical  fatal  attack  of  the  disease. 

Symptoms  in  the  Dog. — The  symptoms  of  rabies  in  the  dog  are 
described  by  Hart  as  follows : 

"The  symptoms  are  generally  given  for  two  different  types,  the 


SYMPTOMS  IN  THE  DOG  601 

furious  or  irritable,  and  the  dumb  or  paralytic.  The  latter  type  is 
always  seen  in  the  terminal  stages  of  the  former;  and  when  the  cases 
are  of  the  dumb  form  from  the  outset  it  is  probable  that  the  toxemia 
is  overwhelming,  and  such  cases  usually  run  a  more  rapid  fatal  course. 

"The  Furious  Type. — In  the  furious  type,  following  the  variable 
period  of  incubation,  there  is  first  noticed  a  change  in  the  dispo- 
sition of  the  animal  which  should  at  once  excite  suspicion.  Playful 
animals  become  morose  and  quiet,  and  reserved  dogs  may  become 
unusually  affectionate.  The  animal  is  nervous  and  easily  excited, 
but  obeys  any  command  of  its  owner.  In  the  course  of  a  day  or  two 
the  nervous  condition  increases  and  the  animal  becomes  irritable 
and  may  snap  if  approached  suddenly  or  startled.  The  bark  becomes 
changed  to  a  long-drawn-out  combination  between  a  whine  and  a 
howl,  impossible  to  describe,  but  never  forgotten  when  once  heard. 
Some  dog  owners  speak  of  it  as  being  somewhat  of  the  nature  of  the 
bark  of  a  foxhound  while  in  the  hunt,  but  this  does  not  properly 
describe  it.  The  animal  if  loose  may  pick  up  and  swallow  straw, 
sticks,  stones,  leather,  and  other  foreign  bodies." 

In  some  cases  there  is  a  tendency  to  bite  parts  of  the  skin,  usually 
at  the  point  where  the  animal  was  bitten,  and  in  one  case  observed 
by  Hart  the  animal  chewed  the  skin  over  the  os  calcis  until  the  entire 
head  of  the  bone  was  exposed  to  view. 

"There  is  a  marked  tendency  in  these  early  stages  for  the  animal 
to  seek  quiet  spots  and  to  hide  in  corners  or  dark  places.  If  an 
attempt  is  made  to  remove  the  animal,  the  person  is  in  great  danger 
of  being  bitten.  The  restlessness  of  the  animal  becomes  more  marked. 
He  may  stand  looking  intently  into  space  as  if  at  an  imaginary  object. 
There  is  difficulty  in  swallowing,  and  saliva  may  dribble  from  the 
mouth.  The  irritability  increases  until  the  animal  becomes  furious, 
biting  at  a  stick  or  other  object  thrust  toward  him.  At  this  stage, 
if  the  animal  is  not  secured,  he  may  leave  home  and  travel  for  miles. 
During  the  long  journey  he  will  fight  with  dogs  and  attack  other 
animals  in  his  path,  but  never  barks  or  makes  any  outcry  during 
these  attacks.  The  animal  may  go  twenty  to  twenty-five  miles  from 
home,  but  always  returns,  if  not  prevented,  in  an  exhausted  condition, 
covered  with  wounds  and  dirt  and  greatly  emaciated.  Signs  of 
commencing  paralysis  now  appear,  with  drooping  of  the  lower  jaw, 
inability  to  swallow,  and  irregularity  in  the  pupils.  The  legs  become 
paralyzed  and  the  animal  passes  into  the  dumb  form  of  the  disease. 

"Dumb  Rabies. — This  form  of  the  disease  occurs  in  only  a  small 
percentage  of  the  cases.  The  symptoms  are  somewhat  similar  to 
those  of  furious  rabies,  except  that  marked  irritability  is  absent  and 
there  is  an  early  appearance  of  paralysis.  This  form  of  the  disease, 
therefore,  renders  the  dog  less  dangerous  than  the  furious,  type. 
The  animal  lies  quietly  in  some  secluded  place  and  appears  to  be 
stupid.  The  paralysis  of  the  jaw  comes  on  early,  the  tongue  pro- 
trudes and  becomes  congested  and  covered  with  dirt,  giving  rise  to 


602  RABIES  AND  THE  NEGRI  BODIES 

the  term  'black  tongue/  which  is  used  in  some  localities,  especially 
in  the  South,  and  a  bad  synonym  for  this  form  of  the  disease.  The 
use  of  the  term  should  be  discouraged,  as  it  tends  to  confound  the 
disease  with  dog  distemper.  The  hind  legs,  trunk,  and  forelegs 
become  paralyzed,  and  death  usually  ensues  in  about  three  days 
while  the  furious  type  lasts  from  six  to  eight  days. 

"Recovery  from  rabies  in  the  dog  after  well-marked  symptoms  have 
developed  is  possible,  and  authentic  cases  have  been  reported  by 
Pasteur,  Roux,  Babes,  Courmont,  Ligniere,  and  Remlinger.  This  is 
so  rare,  however,  that  it  is  of  little  importance  except  in  cases  where 
a  person  has  been  bitten  by  a  dog  showing  all  the  symptoms  of  rabies 
and  the  animal  afterward  recovered.  The  saliva  in  such  cases 
remains  virulent  for  several  days  or  a  week  after  the  subsidence 
of  symptoms,  and  a  diagnosis  can  be  made  by  inoculating  rabbits 
with  some  of  the  salivary  secretion." 

The  consumptive  form  of  the  disease  mentioned  above  develops 
sometimes  after  the  inoculation,  natural  or  artificial,  of  a  virus  of  a 
low  degree  of  virulency.  This  form,  perhaps,  is  a  more  or  less  pure 
toxemia,  characterized  by  slowly  progressing  emaciation,  marasmus, 
and  finally  death  after  complete  exhaustion. 

Symptoms  in  Man. — When  persons  are  bitten  by  rabid  dogs,  if  the 
wounds  are  not  too  lacerated,  and  if  they  are  cleansed  and  treated 
in  the  proper  antiseptic  manner  and  burnt  out  with  the  actual  cautery, 
they  generally  heal  rapidly  and  do  not  give  rise  to  any  special  local 
manifestations,  except  that  there  is  occasionally  a  good  deal  of  itching. 
There  are  no  special  symptoms  during  the  period  of  incubation 
except  that  there  may  be  more  or  less  depression  on  account  of 
apprehension.  When  the  first  symptoms  of  the  stage  of  excitement 
are  about  to  become  manifest  there  may  be  a  good  deal  of  irritation 
at  the  site  of  the  already  healed  wound,  with  lancinating  pains  shooting 
out  from  it;  a  slight  tremor  may  be  present  in  an  extremity  if  it  has 
been  the  seat  of  the  bite.  The  voice  becomes  changed,  it  is  not  clear 
but  slightly  hoarse.  The  patient  becomes  decidedly  depressed  and 
irritable.  Fever  then  generally  occurs,  and  the  disease  progresses 
rapidly  to  its  typical  picture.  The  laryngeal  symptoms  become  more 
severe,  there  is  difficulty  in  respiration,  the  face  becomes  drawn,  and 
shows  an  expression  of  great  anxiety.  Intense  thirst  compels  the 
patient  to  make  strong  efforts  at  drinking,  and  these  bring  about 
contraction  of  the  muscles  of  the  larynx,  so-called  spasm  of  the 
glottis,  with  great  air  hunger  or  dyspnea.  Salivation  becomes 
marked  and  all  reflexes  are  accentuated,  so  that  a  slight  irritation 
may  bring  about  attacks  of  muscular  contractions.  The  patient, 
while  very  thirsty,  is  afraid  to  take  water,  and  the  mere  thought  of 
it  may  bring  about  spasm  of  the  muscles  of  deglutition  and  of  the 
larynx.  As  the  disease  progresses  farther  the  convulsions  affect  more 
and  more  muscles,  including  the  respiratory  muscles  of  the  thorax. 
Patients  now  often  suffer  from  hallucinations  and  delirium,  and 


DIAGNOSIS  603 

maniacal  excitement  may  set  in.  This  stage  of  excitement  lasts 
from  one  to  a  few  days,  and  then  the  paralytic  stage  sets  in;  the  patient 
loses  consciousness,  goes  down  rapidly,  and  dies.  The  stage  of 
excitement  in  man,  as  in  dogs,  may  be  very  short,  not  well  marked 
at  all,  and  then  the  paralytic  state  develops  almost  immediately  after 
the  slight  prodromal  symptoms;  this  form  of  the  disease  is  known 
as  paralytic  rabies  or  dumb  rabies  in  dogs.  Death  in  man  from  rabies 
generally  occurs  two  to  five  days  after  the  outbreak,  or  the  terrible 
struggle  may  be  prolonged  to  eight  to  ten  days. 

Pathologic  Lesions. — There  are  no  characteristic  anatomic  lesions 
from  which  a  diagnosis  of  rabies  in  dogs  or  other  animals  could  be 
made  solely  upon  postmortem  examination  without  the  proper  micro- 
scopic examination.  Dogs  dead  from  rabies  generally  show  the 
paralysis  of  the  muscles  of  the  lower  jaw,  which  hangs  down  and  is 
not  firmly  closed  as  is  generally  the  case  in  consequence  of  rigor 
mortis.  The  stomach  in  dogs  and  other  carnivora  frequently  contains 
instead  of  remnants  of  food,  undigestible  foreign  substances,  such  as 
wood,  coal,  pebbles,  hay,  straw,  leather,  etc.  The  mucosa  is  strongly 
congested,  swollen,  sometimes  superficially  ulcerated  and  hemorrhagic 
at  the  free  margin  of  the  folds.  Foreign  bodies  are  sometimes  found  in 
the  esophagus  or  in  the  intestines.  However,  the  presence  of  foreign 
bodies  in  the  gastro-intestinal  tract  varies  and  their  absence  does  not 
at  all  exclude  a  diagnosis  of  rabies.  Mortley  Axe  made  postmortem 
examinations  of  200  rabid  dogs.  He  found  foreign  bodies  in  90  per 
cent,  of  the  cases,  but  Galtier,  in  1 304  autopsies  on  rabid  dogs  found 
foreign  bodies  in  the  stomach  in  only  657  cases.  The  bladder  is 
usually  empty  or  contains  a  small  amount  of  urine  only,  which  generally 
contains  glucose.  Sometimes  the  bronchial  mucosa  is  hyperemic, 
and  the  salivary  glands  are  frequently  swollen.  The  internal  organs, 
such  as  the  liver,  spleen,  and  kidneys,  are  congested  and  sometimes 
show  evidences  of  parenchymatous  degeneration.  The  cerebrospinal 
meninges  are  edematous  and  hyperemic;  the  gray  matter  on  section 
shows  many  bleeding  points,  indicating  its  congestion.  Hart  states 
that  he  has  seen  quite  a  number  of  cases  of  rabies  in  dogs  where  the 
postmortem  examination  showed  the  presence  of  food  in  the  stomach 
and  a  normal  mucous  membrane. 

Diagnosis. — Changes  in  Nerve  Ganglia. — For  a  number  of  years 
attempts  have  been  made  to  find  changes  in  the  central  nervous 
system  that  would  give  a  reliable  diagnosis  of  rabies.  Babes  was  the 
first  to  describe  certain  characteristic  lesions.  Schaffer,  in  6  cases 
of  hydrophobia  in  man,  found  in  that  part  of  the  spinal  cord  where 
the  nerves  arise  which  go  to  the  region  where  infection  took  place, 
also  in  the  anterior  gray  horns,  around  the  central  canal,  along  the 
neuroglia  bands  of  the  white  matter,  in  the  perivascular  lymph 
channels  and  in  the  vessel  walls  themselves,  inflammatory  round-cell 
infiltration  and  hemorrhages,  both  large  and  capillary.  He  also  found 
in  these  regions  hyaline  degeneration  of  ganglion  cells  and  vacuo- 


604 


RABIES  AND  THE  NEGRI  BODIES 


lation.  A  more  important  observation,  which  for  a  time  gained  great 
importance  in  the  microscopic  diagnosis  of  rabies,  was  made  by  Van 
Gehuchten  and  Nelis.  They  found  in  the  cerebrospinal  and  sym- 
pathetic ganglia,  particularly  in  the  plexus  nodosus  of  the  pneumo- 
gastric  nerve  and  in  the  upper  cervical  ganglia  of  the  sympathetic  nerve, 
a  proliferation  of  the  endothelial  cells  of  the  capsules  of  the  ganglia. 
These  proliferating  cells  invade  the  nerve  cells  of  the  ganglion,  destroy 
them  more  or  less,  and  may  take  their  place  entirely.  The  prolifer- 
ating cells  also  infiltrate  the  periganglionic  region.  The  observations 
of  Van  Gehuchten  and  Nelis  were  soon  confirmed  by  a  number  of 
observers,  among  others  by  Frothingham,  wrho  considers  these 
findings  as  fairly  accurate  means  to  the  diagnosis  of  rabies.  He 
always  found  them  present  in  cases  of  rabies  and  also  in  one  case, 
that  of  a  dog,  where  inoculations  of  emulsified  central  nervous  material 
failed  to  produce  rabies  in  the  injected  animals. 


FIG.  212 


FIG.  213 


'Negri  bodies  in  nerve  cells." 
(After  Wolbach.) 


X  2000. 


"Negri  bodies  in  nerve  cells."     X  1000. 
(After  Wolbach.) 


Today  the  microscopic  diagnosis  of  rabies  is  made  upon  the  finding 
of  the  so-called 

Negri  Bodies. — Negri,  in  1903,  described  certain  inclusions  in 
the  ganglion  cells  of  the  central  nervous  system.  He  claimed  that 
they  were  protozoa  and  the  cause  of  rabies  and  the  most  reliable 
means  of  diagnosticating  this  disease  in  dogs,  other  animals,  and  man. 
These  cell  inclusions,  now  universally  known  as  the  Negri  bodies, 
are  contained  in  the  protoplasm,  particularly  of  the  large  ganglion 
cells  of  certain  regions  of  the  brain.  They  vary  from  minute  dots  to 
large  bodies  of  25  micra  in  diameter.  Negri's  observations  were 
soon  confirmed  by  numerous  observers,  and  it  can  be  stated  today 
that  these  bodies  are  found  almost  without  exception  in  all  cases  of 
natural  rabies  infection,  so-called  street  rabies.  However,  after  the 
virus  has  been  passed  a  number  of  times  through  rabbits  by  subdural 


PLATE  XIV 


h 

S   i 

j 


JS 


??W*'V 
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1.  Various  division  forms  of  the  negri  bodies  (Giemsa  stain).  2.  Smear  of  Ammon's  horn  of  dog, 
showing  negri  bodies  (fi)  stained  red  in  the  large  blue-stained  nerve  cells;  JV,  nucleus  of  nerve  cell.' 
(Van  Gieson's  fuchsin  and  methylene-blue  stain.)  (Park.) 


DIAGNOSIS  605 

inoculation,  that  is,  when  the  virus  has  become  of  the  fixed  type, 
only  the  smallest  bodies  are  seen.  The  classification  of  these  bodies, 
to  which  Williams  has  given  the  zoological  name  neuroryctes  hydro- 
phobia, cannot  yet  be  regarded  as  established,  and  Calkins  states 
that  we  are  not  yet  justified  in  classifying  them  as  sporozoa,  because 
their  variable  form,  their  uninucleate  condition,  leading  to  chromatin 
distribution  and  budding,  though  found  in  other  protozoa,  are  not 
characteristic  for  sporozoa.  Williams  and  Lowden,  whose  careful 
researches  of  the  Negri  bodies  have  contributed  much  to  our  know- 
ledge, describe  them  as  follows:  "They  measure  in  size  from  0.5 


FIG.  214 


* 

I 


10. 


» 
• 


"Negri  bodies,"  or  Neuroryctes  hydrophobias,  in  different  stages  of  chromatin  distribution. 

(After  Negri.) 

to  18  micra  (Negri  saw  some  as  large  as  25  micra);  no  very  large 
forms  are  found  in  the  early  stages  of  the  disease,  and  in  the  fixed 
virus  infection  only  the  very  small  forms  are  found.  They  vary 
much  in  shape,  from  mere  irregular  points  to  larger,  round,  oval, 
or  elongated  bodies.  They  are  generally  inside  of  the  large  ganglion 
cells,  but  in  smear  preparations  (see  below)  they  may  be  found 
squeezed  out  of  the  cells.  Whatever  the  variety  of  species  of  animals 
infected  the  bodies  preserve  their  same  general  characteristic  structure, 
namely,  a  hyaline  cytoplasm  with  an  entire  margin,  and  with  one  or 


606  RABIES  AND  THE  NEGRI  BODIES 

more  inner  bodies  having  a  more  or  less  complicated  and  regular 
structure."  Negri  early  recognized  that  the  rabies  bodies  in  the  gan- 
glion cells  of  the  brain  had  a  nucleus,  and  he  later  stated  that  their 
chromatin  had  either  a  solid  or  a  reticular  structure,  while  their 
cytoplasm  contained  a  variable  number  of  chromatin  granules.  The 
latter  are  frequently  arranged,  as  stated  by  Williams  and  Lowden,  in 
a  more  or  less  complete  circle  about  the  nucleus.  They  are  somewhat 
irregular  in  outline  and  size,  being  occasionally  ring-shaped,  some- 
times elongated,  often  in  twos,  due  probably  to  the  ative  changes 
of  growth  and  division.  The  difference  in  shape  of  the  Negri  bodies, 
such  as  round,  triangular,  etc.,  is  due  to  their  position  in  the  ganglion 
cells,  since  their  bodies  are  very  plastic  and  easily  adaptable  to  a 
variety  of  positions.  The  variability  in  shape  is  also  probably  largely 
due  to  a  rapid  multiplication.  The  division  forms  suggest  rapid 
growth  and  multiplication.  The  elongated  forms,  containing  from 
two  to  five  or  even  six  nuclei,  are  the  result  of  rapid  nuclear  division 
without  corresponding  cell  division.  This  condition  is  found  quite 
frequently  in  protozoa.  The  elongation  of  the  protoplasm  is  probably 
due  to  the  position  of  these  bodies  between  the  nerve  fibrils,  and  to 
their  great  plasticity. 

Under  the  most  favorable  conditions  (fixed  virus),  growth  and 
division  occur  most  rapidly  and  simply,  the  tiny  forms  dividing  and 
redividing  apparently  indefinitely.  Small  mulberry  masses  are  found 
during  this  stage,  but  whether  they  are  the  result  of  the  breaking  up 
of  a  larger  form  or  of  the  rapid  division  of  a  tiny  form  it  is  impossible 
as  yet  to  say. 

In  cases  where  there  has  been  an  inoculation  of  comparatively 
small  quantities  of  the  virus,  i.  e.,  a  small  number  of  forms  of  the 
parasite  capable  of  immediate  infection,  or  in  cases  where  there  has 
been  an  infection  of  less  susceptible  animals  (dogs,  cattle,  human 
beings,  etc.),  or  with  a  less  accustomed  virus  (fixed  virus  of  rabbits 
into  guinea-pigs  or  mice),  a  slower  growth  is  obtained  with  its  larger 
structures  and  different  division  forms.  The  chromatin  accumu- 
lation in  the  form  of  a  definite  nucleus  apparently  undergoes  frag- 
mentation very  easily,  resulting  in  forms  containing  two  to  several 
central  bodies,  some  rounded,  some  elongated,  some  of  unequal 
division  similar  to  budding.  Then  forms  are  found  with  bodies 
apparently  differentiated  within  one  membrane,  and  bodies  with 
practically  all  stages  of  hour-glass  constriction,  indicating  transverse 
division.  Many  pairs,  unequal  in  size,  apparently  fusing  or  dividing, 
have  been  seen,  and  finally,  large  bodies  with  the  chromatin  scattered 
throughout  the  whole  organism  in  the  form  of  tiny,  unevenly  rounded, 
or  elongated  masses.  One  or  two  will  be  larger,  indicating  the  remains 
of  the  nucleus.  In  these  forms  are  found  all  stages  of  apparent 
budding,  varying  somewhat  in  size,  some  being  very  tiny.  The 
formation  of  buds  accounts  for  the  appearance  in  the  same  cell  of 
both  large  and  small  forms.  It  also  helps  to  account  for  the  rapid 


DIAGNOSIS  607 

spread  of  the  organisms.  These  tiny  budded  forms  similar  to  "swarm 
spores"  are  probably  motile  and  pass  quickly  to  other  host  cells. 

The  Rapid  Microscopic  Diagnosis  of  Rabies. — Williams  and  Lowden 
worked  out  a  rapid  method  of  diagnosticating  microscopically  rabies, 
and  an  almost  identical  method  by  Frothingham  was  published  about 
the  same  time. 

The  author  has  used  their  smear  method  since  the  fall  of  1906  and 
has  found  it  very  satisfactory  and  reliable,  and  it  is  highly  recom- 
mended in  all  cases  where  a  rapid  microscopic  diagnosis  is  necessary. 
The  steps' of  Williams'  and  Lowden's  method  are  as  follows: 

1.  Glass  slides  and  cover-glasses  are  washed  thoroughly  with  soap 
and  water,  then  heated  in  the  flame  to  get  rid  of  greasy  substance. 

2.  A  small  bit  of  the  gray  substance  of  the  brain  to  be  examined  is 
cut  out  with  a  small,  sharp  pair  of  scissors  and  placed  on  the  slide 
about  one  inch  from  the  end,  so  as  to  leave  enough  room  for  a  label. 
The  cut  in  the  brain  should  be  made  at  right  angles  to  its  surface  and 
a  thin  slice  taken,  avoiding  the  white  matter  as  much  as  possible. 

3.  A  cover-slip  placed  over  the  piece  of  tissue  is  pressed  upon  it 
until  the  brain  substance  is  spread  out  in  a  moderately  thin  layer, 
then  the  cover-slip  is  moved  slowly  and  evenly  over  the  slide  to  the 
end  opposite  the  label.    Only  slight  pressure  should  be  used  in  making 
the  smear,  but  slightly  more  should   be  exerted  on  the  cover-glass 
toward  the  label  side  of  the  slide,  thus  allowing  more  of  the  nerve 
tissue  to  be  carried  farther  down  the  slide  and  producing  better 
spread  nerve  cells.     If  any  thick  places  are  left  at  the  edge  of  the 
smear,  one  or  two  of  them  may  be  spread  out  toward  the  side  of 
the  slide  with  the  edge  of  the  cover-glass.     If  the  first  smear  does 
not  turn  out  successfully  others  should  be  made  until  a  satisfactory 
one  is  obtained. 

4.  For  diagnosis  work  such  a  smear  should  be  made  from  at  least 
three  different  parts  of  gray  matter  of  the  central  nervous  system: 
First,  from  the  cortex  in  the  region  of  the  fissure  of  Rolando  or  in 
the  region  corresponding  to  it  (in  the  dog  the  convolution  around 
the  crucial  sulcus);  second,  from  Ammon's   horn;   third,  from  the 
cerebellum. 

5.  The  smears  are  dried  in  air  and  subjected  to  one  or  both  of  the 
following  staining  methods : 

(a)  Giemsa's  Solution. — The  smears  are  fixed  in  methyl  alcohol 
(commercial  is  just  as  good  as  pure)  for  about  five  minutes.  The 
staining  solution  recommended  last  by  Giemsa  is  as  follows:  1  drop 
of  the  stain  to  every  c.c.  of  distilled  water  made  alkaline  by  the 
previous  addition  of  1  drop  of  a  1  per  cent,  solution  of  potassium 
carbonate  to  10  c.c.  of  water.  This  is  poured  over  the  slide  and 
allowed  to  stand  from  one-half  to  three  hours.  The  longer  time 
brings  out  the  structure  better,  and  in  twenty-four  hours  well-made 
smears  are  not  overstained.  After  the  stain  is  poured  off  the  smear 
is  washed  in  running  tap  water  for  one  to  three  minutes,  and  dried 


608  RABIES  AND  THE  NEGRI  BODIES 

with  filter  paper.  If  the  smear  is  thick  the  "bodies"  may  come  out  a 
little  more  clearly  by  dipping  in  50  per  cent,  methyl  alcohol  before 
washing  in  water,  then  the  washing  need  not  be  as  thorough.  By 
this  method  of  staining  the  cytoplasm  of  the  "bodies"  stains  blue  and 
the  central  bodies  and  chroma toid  granules  stain  a  blue  red  or  azure. 
Generally  the  larger  "bodies"  are  a  darker  blue  than  the  smaller;  the 
smallest  of  all  may  be  very  light.  The  stain  varies  somewhat  according 
to  the  thickness  of  the  smear.  Some  have  a  robin's  egg  blue  tint,  but 
this  is  due  to  long  fixation  in  the  methyl  alcohol.  In  this  case  the 
red  blood  cells  may  have  a  greenish  tint.  The  cytoplasm  of  the  nerve 
cells  stains  blue  also,  but  with  a  successfully  made  smear  the  cytoplasm 
is  so  spread  out  that  the  outline  and  structure  of  most  of  the  "bodies" 
are  seen  distinctly  within  it.  The  nuclei  of  the  nerve  cells  are  stained 
red  with  the  azure,  the  nucleoli  a  dull  blue,  the  red  blood  cells  a  pink 
yellow,  more  pink  if  the  decolorization  is  used.  The  "bodies"  have 
an  appearance  of  depth,  due  to  their  slightly  refractive  qualities. 

For  diagnostic  purposes  this  method  of  staining  may  be  shortened 
as  follows:  Methyl  alcohol,  five  minutes,  equal  parts  of  the  Giemsa 
solution  and  distilled  water,  ten  minutes.  In  this  way  "bodies"  are 
generally  brought  out  well  enough  for  diagnosis,  and  sometimes  the 
structure  shows  distinctly.  It  is  always  well,  however,  to  make  smears 
enough  for  the  longer  method  of  staining,  in  case  the  shorter  should 
prove  unsatisfactory. 

(6)  The  eosin-methylene  blue  method  recommended  by  Mallory. 
The  smears  are  fixed  in  Zenker's  solution  for  one-half  hour;  after 
being  rinsed  in  tap  water  they  are  placed  successively  in  95  per  cent, 
alcohol  +  iodin,  one-quarter  hour;  95  per  cent,  alcohol,  one-half 
hour;  absolute  alcohol,  one-half  hour;  saturated  watery  eosin  solution, 
twenty  minutes,  rinsed  in  tap  water;  alkaline  methylene-blue  solution, 
fifteen  minutes;  differentiated  in  95  per  cent,  alcohol  lasting  from  one 
to  five  minutes,  and  dried  with  filter  paper.  With  this  method  of 
staining  the  cytoplasm  of  the  "bodies"  is  a  magenta,  light  in  the 
small  bodies,  darker  in  the  larger;  the  central  bodies  and  chromatoid 
granules  are  very  dark  blue,  the  nerve  cytoplasm  a  light  blue,  the 
nucleus  a  darker  blue,  and  the  red  blood  cells  a  brilliant  eosin  pink. 
With  more  decolorization  in  the  alcohol  the  "bodies"  are  not  such  a 
deep  magenta  and  the  difference  in  color  between  them  and  the  red 
blood  cells  is  not  so  marked. 

The  "bodies"  and  the  structure  are  often  more  clearly  defined  with 
this  method,  and  perhaps  on  the  whole  it  is  better  to  use  it  for  making 
diagnoses;  but  when  there  are  only  tiny  "bodies"  present,  or  when 
the  brain  tissue  is  old  and  soft,  the  Giemsa  stain  seems  to  be  the 
more  successful.  Above  all,  when  one  wishes  to  study  the  nature  of 
the  central  structures  and  granules  the  Giemsa  stain  must  be  used. 
Both  methods  are  strongly  recommended. 

The  technique  of  the  section  work  is  as  follows:  (1)  The  small 
pieces  are  left  in  Zenker's  fluid  for  three  to  four  hours;  (2)  washed 


DIAGNOSIS  609 

in  tap  water  for  five  minutes;  (3)  placed  in  80  per  cent,  alcohol  + 
iodin  (enough  tincture  of  iodin  added  to  give  port-wine  color)  for 
about  twenty-four  hours;  (4)  95  per  cent,  alcohol  +  iodin,  twenty- 
four  hours;  (5)  95  per  cent,  alcohol,  twenty-four  hours;  (6)  absolute 
alcohol,  from  four  to  six  hours;  (7)  cedar  oil  until  cleared;  (8)  cedar 
oil  +  paraffin,  52°,  aa,  two  hours ;  (9)  paraffin,  52°,  two  hours  in  each 
of  two  baths;  (10)  boxing;  (11)  sections  are  cut  at  3  to  6  micra,  dried 
in  thermostat  at  36°  C.  for  about  twenty-four  hours,  protected  from  the 
dust,  and  stained  according  to  the  eosin-methylene-blue  method  of 
Mallory.  The  important  point  in  the  technique  is  the  time  the 
material  is  allowed  to  remain  in  Zenker.  According  to  the  author's' 
experience  two  hours'  fixation  is  not  enough,  three  or  four  hours  is 
very  good,  and  with  every  hour  after  five  hours  the  result  becomes 
less  satisfactory.  Left  in  Zenker  overnight  the  tissue  is  granular 
and  takes  the  eosin  stain  more  or  less  deeply,  both  of  which  results 
interfere  with  the  appearance  of  the  tiniest  "bodies,"  especially  of 
the  very  delicate,  tiny  forms  found  in  sections  from  fixed  virus. 
Another  point  in  favor  of  the  short  fixation  in  Zenker  is  that  the 
precipitate  formed  by  the  mercury  is  not  so  great,  and  is  more  easily 
eliminated,  which  is  a  very  great  help  in  the  identification  of  the 
tiniest  forms. 

For  routine  work  for  diagnostic  purposes  the  method  of  fixing 
the  smears  in  Zenker's  solution  and  staining  subsequently  by  the 
eosin-methylene-blue  method  of  Mallory  is  the  simplest  and  most 
reliable. 

Methods  of  Preparing  Material  for  Laboratory  Examination. — Few 
veterinarians  are  so  situated  and  trained  that  they  can  make  a  depend- 
able microscopic  diagnosis  of  rabies  in  a  dog.  However,  if  a  sus- 
pected animal  has  bitten  other  dogs  or  cattle,  horses,  persons,  etc.,  a 
definite  diagnosis  should  always  be  made,  so  that  the  proper  measures 
can  be  taken  if  the  animal  did  suffer  from  rabies.  One  of  the  worst 
things  to  do  if  a  suspicious  dog  has  bitten  a  person  is  to  kill  the  dog 
at  once,  because  in  the  very  earliest  stages  of  rabies  it  may  be  impos- 
sible to  find  the  Negri  bodies,  yet  the  dog  may  have  had  rabies  and  its 
saliva  may  already  have  been  infective.  The  proper  thing  to  do  is  to 
isolate  and  safely  detain  such  a  dog  and  to  watch  for  the  development 
of  symptoms.  If  the  animal  sickens  under  typical  symptoms  of  rabies 
it  may  be  killed  and  the  brain  examined  microscopically,  or  if  the 
symptoms  are  not  clear  it  may  be  allowed  to  die  and  then  the  brain 
should  be  examined.  The  veterinarian  in  charge  of  the  case  should 
make  a  postmortem  examination  on  the  dog  and  the  head  should  be 
severed  from  the  body  somewhere  in  the  middle  of  the  cervical  ver- 
tebrae, so  that  the  upper  cervical  ganglia  are  left  undisturbed,  that 
they  may,  if  desirable,  be  included  in  the  microscopic  examination. 
The  head  should  then  be  wrapped  in  a  piece  of  cheese  cloth,  or,  if 
available,  in  a  thin  rubber  sheet,  as  used  in  surgical  dressings,  placed 
in  a  tin  bucket,  then  in  a  wooden  box,  and  forwarded  by  express 
39 


610  RABIES  AND  THE  NEGRI  BODIES 

to  the  laboratory  where  the  examination  is  to  be  made.  During  the 
warm  season  the  tin  bucket  should  be  surrounded  by  cracked  ice. 
These  measures,  however,  are  sufficient  only  when  the  package  can 
reach  its  destination,  particularly  in  summer,  in  less  than  twenty-four 
hours.  If  it  has  to  be  on  the  road  longer,  or  if  the  weather  is  very 
warm,  it  is  better  to  take  the  brain  out  of  the  cranium.  The  entire 
brain  can  then  be  placed  in  pure  neutral  glycerin  and  sent  to  the 
laboratory.  This  method  has  been  frequently  recommended,  but  it 
is  almost  impossible  to  find  the  Negri  bodies  in  a  brain  that  has 
been  preserved  in  glycerin.  The  following  method  is,  therefore,  better : 
Take  out  the  brain,  divide  into  two  equal  halves.  Place  one  half  into 
several  times  its  bulk  of  pure  neutral  glycerin,  the  other  half  into  a 
strong  formalin  solution  (1  part  of  formalin  to  4  parts  of  water). 
When  the  brain  arrives  at  the  laboratory,  very  small  pieces,  not  more 
than  1  square  centimeter  large  and  less  than  half  a  centimeter  thick, 
are  cut  out  from  the  gray  matter  of  the  Ammon's  horn,  the  region 
of  the  fissure  of  Rolando  and  the  cortex  of  the  cerebellum  of  the 
formalin  preserved  material.  These  pieces  are  placed  in  Zenker's 
solution  for  a  few  hours,  then  washed  rapidly  in  running  water, 
then  placed  for  one  hour  in  pure  water-free  acetone,  which  is  changed 
once,  and  then  dropped  into  melted  paraffin,  sectioned,  and  stained 
by  the  eosin-methylene-blue  method.  The  fixation  by  Zenker's  fluid 
may  even  be  left  out  and  the  sections  can  then  be  simply  stained  by 
hematoxylin  and  eosin.  The  watery  eosin  solution  should  then  be 
somewhat  stronger  than  when  used  for  ordinary  work  in  histology  or 
pathology.  If  the  Negri  bodies  are  found  the  other  half  of  the  brain 
in  glycerin  is  not  needed,  but  if  they  are  not  found  and  the  investigation 
is  to  be  carried  on,  an  emulsion  in  physiologic  salt  solution  is  made 
from  the  brain  substance  in  glycerin  and  injected  into  rabbits  by  the 
subdural  method.  This  necessitates  opening  the  cranium  by  trephin- 
ing, followed  by  the  subdural  injection.  (Details  of  this  method  of 
inoculation  are  given  below.) 

Are  the  Negri  Bodies  Protozoa? — That  the  Negri  bodies  are  indeed 
protozoa  is  not  accepted  as  an  established  fact  by  all  observers,  and 
some  hold  that  they  are  degenerative  or  other  products  of  elements 
of  the  central  nervous  system  of  the  animal  or  person  infected  with 
rabies.  The  main  reason  for  this  belief  is  the  observation  first  made 
by  Remlinger  and  Riffat-Bey,  and  confirmed  by  others,  that  emul- 
sions of  the  central  nervous  system  frequently  can  be  filtered  through 
Chamberland  and  Berkefeld  filters  without  losing  their  infectiousness. 
However,  it  must  not  be  forgotten  that  there  are  very  small  Negri 
bodies  which  are  probably  very  plastic  and  which  under  pressure  may 
easily  pass  the  pores  of  porcelain  or  clay  filters.  The  preponderance 
of  evidence  today  appears  to  be  in  favor  of  the  protozoan  nature  of 
the  Negri  bodies. 

Peculiar  Biological  Properties  of  Neuroryctes  Hydrophobias. — If  the 
Negri  bodies  are  protozoa,  strict  parasites  which  live  and  multiply  in 


SPREAD  OF  VIRUS  IN  ORGANISM  OF  SUSCEPTIBLE  ANIMALS  611 

the  ganglion  cells  of  the  central  nervous  systems  of  higher  animals, 
they  furnish  one  of  the  most  wonderful  examples  of  how  a  parasite 
in  the  struggle  for  existence,  with  the  survival  of  the  fittest,  may 
develop  those  properties  which  will  insure  its  propagation  from  one 
host  to  another.  To  the  parasitic  tubercle  bacillus  are  open  a  thousand 
and  one  routes  by  which  it  may  be  transferred  from  one  animal  to 
another;  to  the  neuroryctes  hydrophobise,  as  far  as  known,  only  one 
way,  namely,  that  of  an  infected  animal  biting  and  wounding  another. 
There  is  no  disease  which,  particularly  in  carnivora,  leads  to  such  an 
intense,  insane  desire  to  bite  as  does  hydrophobia,  and  this  symptom 
must  be  looked  upon  as  one  due  to  disturbances  in  the  ganglion 
cells  depending  upon  poisons  developed  and  produced  by  the  neuro- 
ryctes for  the  sole  and  special  purpose  of  insuring  the  survival  of 
the  race  after  the  death  of  the  host,  which  it  kills  by  its  own  multi- 
plication. That  the  parasitic  microorganisms  of  rabies  should  produce 
as  one  of  the  very  prominent  symptoms  of  the  disease  the  psychic 
disturbances  which  lead  to  the  intense  desire  to  bite,  particularly 
in  canines,  cats,  rats,  and  occasionally  in  rabbits,  is  the  more  remark- 
able when  it  is  considered  that  the  tetanus  toxin  which  also  directly 
affects  the  central  nervous  system  travels  to  it  along  the  peripheral 
nerves,  produces  symptoms,  as  far  as  convulsions  and  paralyses  are 
concerned,  very  similar  to  the  rabic  virus,  does  not  in  any  way  lead 
to  similar  or  identical  psychic  disturbances.  Tetanus,  however, 
cannot  be  spread  through  the  bite  of  an  infected  animal  and  the 
generally  saprophytic  tetanus  bacillus  does  not  depend  for  the  pre- 
servation of  the  race  upon  the  invasion  of  higher  animals. 

Another  striking  feature  in  the  life  history  of  neuroryctes  hydro- 
phobise is  the  fact  that  while  not  to  any  extent  found  in  other  tissues 
but  those  of  the  central  nervous  system,  it  is  always  present  in  the 
salivary  glands  and  excreted  with  the  saliva  to  thus  insure  its  transfer 
to  a  new  host.  The  mode  of  transfer  from  one  host  to  another  is 
identical  in  principle  with  that  found  in  malaria.  However,  in  this 
disease  the  protozoan  parasites  are  first  taken  up  by  an  intermediary 
host,  the  mosquito,  to  wander  from  its  stomach  into  the  salivary 
glands,  and  to  be  transferred  by  biting  to  its  more  permanent  host, 
man,  birds,  or  other  animals. 

Spread  of  the  Virus  in  the  Organism  of  Susceptible  Animals. — It  has 
been  shown  by  the  experiments  of  Pasteur  and  others  that  the  rabic 
virus  from  its  place  of  entrance  in  the  body  of  a  susceptible  animal 
travels  along  the  peripheral  nerves  to  the  central  nervous  system. 
Rabies  can  always  be  produced  by  injecting  the  virus  directly  into 
the  peripheral  nerves  or  even  by  moistening  with  an  emulsion  the 
central  end  of  a  divided  nerve.  On  the  other  hand,  if  the  peripheral 
end  of  a  cut  nerve  is  infected  and  the  connection  between  this  part 
and  the  central  nervous  system  carefully  destroyed  then  infection 
does  not  occur.  The  most  pronounced  changes  in  the  cord  in  rabies 
are  always  found  in  that  part  which  supplies  the  peripheral  nerves 


612  RABIES  AND  THE  NEGRI  BODIES 

to  the  region  where  the  infection  by  bites  occurred.  Hellman  and 
Marx  have  shown  that  if  virus  is  carefully  injected  into  the  peritoneal 
cavity  in  such  a  manner  that  nerves  are  not  injured,  infection  does 
not  occur.  Intravenous  injection  leads  to  infection  in  dogs  and  rabbits 
but  not  in  herbivora,  which  shows  that  the  possibility  of  infection 
through  the  blood  current  cannot  be  denied,  but  everything  points  to 
the  peripheral  nerves  as  the  common  routes  by  which  the  virus  usually 
travels  to  the  central  nervous  system. 

Immunization  in  Rabies. — The  first  attempts  to  immunize  animals 
were  made  in  1881  by  Gaultier,  who  injected  saliva  from  rabid  dogs 
into  the  jugular  veins  of  a  number  of  sheep  and  one  horse.  This 
procedure  did  not  produce  rabies  in  the  animals  so  treated,  and, 
according  to  the  claim  of  the  experimenter,  protected  them  against 
subsequent  bites  from  hydrophobic  dogs.  Nocard  and  Roux  repeated 
Gaultier's  experiments  on  sheep,  goats,  cattle,  and  horses,  but  instead 
of  using  saliva  they  employed  for  the  intravenous  injections  emulsions 
from  the  central  nervous  system,  and  they  confirmed  the  observation 
of  their  predecessor,  that  such  treatment  produced  immunity  against 
subsequent  intraocular  inoculation  and  against  the  bites  of  rabid 
animals. 

The  Pasteur  Treatment. — Between  the  experiments  of  Gaultier  and 
those  of  Nocard  and  Roux,  Pasteur  had  taken  up  the  work  of  immu- 
nization against  rabies.  He  had  previously  discovered  the  method 
to  immunize  animals  against  fowl  cholera,  hog  erysipelas,  and  anthrax 
by  the  use  of  attenuated  cultures  or  viruses,  and  he  based  his  experi- 
ments on  rabies  from  the  start  upon  attempts  to  prepare  an  attenuated 
virus.  He  first  succeeded  in  obtaining  it  by  repeated  passages  through 
monkeys.  If  a  virus  so  obtained  was  injected  subcutaneously  into 
dogs  it  did  not  produce  hydrophobia  and  protected  the  animals  so 
treated  against  the  subsequent  bites  of  rabid  dogs.  The  method 
on  which  the  so-called  Pasteur  treatment  of  hydrophobia  is  based 
was  subsequently  worked  out  by  Pasteur,  Chamberland,  and  Roux. 
Its  principle  is  the  following:  Rabbits  are  first  inoculated  subdurally 
from  the  virus  obtained  from  dogs  which  have  developed  rabies. 
This  is  the  so-called  street  virus.  It  is  of  variable  virulency  and 
produces  hydrophobia  in  rabbits  after  a  variable  period  of  incubation. 
If  the  virus  is  then  passed  on  from  rabbit  to  rabbit,  always  by  sub- 
dural  inoculation,  the  period  of  incubation  is  more  and  more  shortened 
on  account  of  the  increasing  virulency  of  the  living  poison.  After 
a  number  of  passages  the  virulency  reaches  a  certain  maximum 
beyond  which  it  cannot  be  increased,  and  the  period  of  incubation 
becomes  stationary  at  six  to  seven  days.  The  virus  so  obtained, 
which  also  shows  an  increased  virulency  when  inoculated  subdurally 
into  animals  other  than  rabbits,  is  known  as  the  fixed  virus.  It  can 
be  attenuated  by  a  variety  of  methods,  but  the  method  first  employed 
by  Pasteur  and  still  used  by  many  consists  in  exposing  the  spinal 
cords  of  rabbits  which  contain  the  fixed  virus  to  drying-out  processes. 


THE  PASTEUR  TREATMENT  613 

The  longer  the  drying-out  process  is  allowed  to  go  on  the  more 
attenuated  becomes  the  virus. 

The  emulsion  used  in  inoculating  rabbits  and  also  in  the  protective 
inoculations  is  always  prepared  from  the  cords  of  rabbits  containing 
the  fixed  virus  by  rubbing  up  pieces  of  the  cord  with  sterile  physio- 
logical salt  solution  until  a  milky  emulsion  is  obtained.  It  is,  of  course, 
always  necessary  first  to  start  from  the  cord  of  a  hydrophobic  dog 
and  to  continue  inoculating  rabbits  subdurally  until  a  fixed  virus  has 
been  obtained. 

Technique. — The  technique  of  subdural  inoculation  in  rabbits  is  as 
follows:  The  animal  is  fastened  to  a  small  animal  operating  table, 
resting  on  its  abdomen,  and  the  head  is  fixed  so  that  it  cannot  move.1 
The  scalp  is  then  cut  open  very  near  the  median  line  from  near  the 
posterior  angle  of  the  eye  toward  the  insertion  of  the  ear.  The 
periosteum  is  next  removed  from  the  bone.  The  latter  is  then  per- 
forated by  a  small  trephine,  the  tip  of  which  is  not  more  than  about 
2  to  3  mm.  in  diameter.  The  piece  of  bone  cut  out  is  removed  by  a 
small  hook.  The  dura  mater  then  becomes  visible  and  it  is  perforated 
by  the  fine  needle  of  a  hypodermic  syringe  and  from  i  to  J  c.c,  of 
the  emulsion  is  slowly  injected  into  the  subdural  space.  In  inserting 
the  needle  it  should  be  held  rather  obliquely  and  directed  forward  and 
outward;  in  this  manner  injury  to  the  brain  is  avoided.  After  the 
emulsion  has  been  injected  and  the  needle  withdrawn  the  linear 
incision  of  the  scalp  is  closed  by  sutures  and  a  collodion  dressing  is 
put  on  the  wound.  It  goes  without  saying  that  everything  has  to  be 
done  aseptically  in  order  to  avoid  the  occurrence  of  a  septic  menin- 
gitis which  might  kill  the  experimental  animal  before  rabies  had  time 
to  develop.  This  generally  occurs  nine  to  twenty-one  days  after  the 
inoculation  of  the  street  virus  and  six  to  seven  days  after  the  inoculation 
of  the  fixed  virus.  The  subdural  method  is  used  exclusively  in  the 
preparation  of  the  fixed  virus;  it  is  the  only  reliable  one,  and  it  alone 
should  be  used  in  diagnostic  work.  Even  the  intraocular  method 
is  not  absolutely  reliable,  and  all  other  methods  are  very  unreliable. 
Rabbits  inoculated  with  street  virus  generally  develop  the  picture  of 
dumb  rabies;  however,  this  is  not  invariably  the  case,  and  they 
sometimes  develop  the  furious  type,  become  aggressive,  and  are  liable 
to  bite.  Since  the  cases  where  hydrophobic  rabbits  did  bite  persons 
all  occurred  in  laboratories,  and  since  the  persons  bitten  all  promptly 
received  treatment,  it  is  not  known  whether  such  an  injury  might 
lead  to  an  outbreak  of  hydrophobia. 

Rabbits  after  being  inoculated  with  street  virus,  before  developing 
typical  symptoms,  generally  show,  as  first  noticed  by  Babes,  an 
elevation  of  temperature.  Others  have  denied  that  this  prodromal 
elevation  of  temperature  regularly  occurs.  This  is  followed  by  a 

»  Kraus  states  that  it  is  not  necessary  to  tie  rabbits  to  the  operating  table;  they  can  be  held 
by  an  assistant  and  the  operation  can  be  performed.  This  saves  much  time  if  a  number  of 
animals  are  to  be  inoculated. 


614  RABIES  AND  THE  NEGRI  BODIES 

slight  paralysis  of  the  hind  legs,  so  that  the  animals  can  be  easily 
thrown  over.  They  then  become  restless  and  cramps  of  the  muscles 
of  the  lower  jaw  occur.  The  disease  now  may  take  on  the  furious 
type,  but  more  generally  the  paralyses  become  more  marked,  the 
hind  legs  are  completely  paralyzed,  and  the  front  legs  become  likewise 
affected.  Within  a  few  days  great  emaciation  occurs  and  death 
generally  follows  four  to  five  days  after  the  first  symptoms  became 
manifest.  After  subdural  inoculation  of  the  fixed  virus  the  furious 
type  of  rabies  is  never  developed  in  rabbits. 

Preparation  of  the  Attenuated  Virus. — Where  material  for  the 
Pasteur  treatment  of  rabies  is  prepared  it  is  necessary  to  inoculate 
at  least  two  rabbits  every  day,  so  that  a  complete  series  of  attenuated 
viruses  is  always  on  hand,  even  if  occasionally  one  cord  should 
become  spoiled  by  bacterial  contamination.  The  inoculated  animals 
must  be  kept  separate  from  the  others,  and  they  must  be  carefully 
watched  for  the  development  of  any  other  disease,  which  would  make 
them  unavailable  for  use  for  the  preparation  of  rabies  virus.  The 
inoculated  animals  are  killed  by  cutting  the  throat  and  bleeding, 
about  twenty-four  hours  before  their  expected  death  from  rabies. 
They  are  then  skinned,  the  abdominal  and  thoracic  cavities  are 
opened  and  examined.  If  no  other  pathologic  lesions  but  those  of 
rabies  are  seen  the  animals  are  stretched  out  on  a  sterile  board, 
abdomen  down,  and  the  external  surface  of  the  back  is  thoroughly 
washed  with  a  solution  of  lysol.  The  muscles  of  the  back  are  removed 
from  the  vertebral  column  and  the  latter  is  then  opened  with  special 
scissors  devised  for  the  purpose;  next  the  roots  of  the  spinal  nerves  on 
each  side  are  severed  with  a  fine  knife,  the  dura  mater  is  laid  open, 
and  the  upper  part  of  the  cord  is  tied  with  a  piece  of  sterile  silk 
or  grasped  with  a  small,  sterile  platinum  hook.  The  cord  is  then 
lifted  out  and  cut  in  the  middle  of  its  course.  >  The  upper  piece  is 
at  once  placed  in  a  wide-mouthed  sterile  flask  and  so  suspended 
that  it  does  not  touch  the  walls  of  the  vessel.  Next  a  small  portion 
of  the  lower  part  is  cut  off  and  dropped  into  a  sterile  Petri  dish  or 
other  sterile  dish  for  subsequent  examination,  as  to  the  absence  of 
contamination  by  bacteria.  Then  the  small  lower  portion  is  removed 
and  treated  like  the  upper  portion.  The  bottles  in  which  the  pieces 
of  cord  are  suspended  are  of  special  construction  and  contain  caustic 
soda  which  is  used  to  absorb  water  and  bring  about  the  drying  out 
of  the  material.  The  flasks  with  the  pieces  of  cord  are  kept  in  a 
perfectly  dark  place  where  the  temperature  is  stationary  at  20°  C. 
After  the  cord  has  been  taken  care  of  the  internal  organs  of  the 
abdominal  and  thoracic  cavities  and  the  brain  are  carefully  dissected 
to  show  that  they  are  free  from  pathologic  lesions  which  would  bar 
the  material  from  use.  When  the  pieces  of  cord  have  gone  through 
the  drying-out  process  they  are  rubbed  up  and  emulsified  with  sterile 
bouillon,  or,  still  better,  with  Babes'  fluid,  consisting  of  sulphate  of 
sodium,  5  gr.;  chloride  of  sodium,  6  gr.,  and  water  enough  to  make 


CORD  LESIONS  AFTER  THE  PASTEUR  TREATMENT       615 

1000  c.c.  One  gram  of  cord  is  rubbed  up  with  5  c.c.  of  fluid.  Of 
this  emulsion  1  to  3  c.c.  are  used  on  persons  which  receive  the  pre- 
ventive Pasteur  treatment,  the  dose  varying  as  to  the  severity  of 
the  case  and  as  to  the  age  of  the  patient.  Pasteur  devised  three 
methods  which  are  still  practised:  one  for  cases  of  light  wounds 
which  came  to  treatment  immediately,  the  two  others  for  more  severe 
cases  and  for  later  treatments.  In  all  cases  treatment  is  begun  with 
an  emulsion  from  a  cord  which  has  been  subjected  to  the  drying-out 
process  for  fourteen  days,  next  a  cord  is  used  which  has  been  dried 
out  for  thirteen  days,  and  so  on,  until  finally  a  cord  is  used  which 
has  been  kept  in  a  bottle  over  caustic  soda  for  three  days.  The 
whole  course  of  treatment  lasts  from  eighteen  to  twenty-one  days, 
because  the  same  type  of  emulsion  is  used  on  two  consecutive  days  at 
times.  Wounds  of  the  face  are  always  treated  by  the  most  energetic 
method,  which  is  carried  out  for  twenty-one  days. 

The  original  method  of  Pasteur  has  been  modified  by  a  number 
of  authors.  Hoegys  uses  fresh  virus  obtained  from  the  medulla  of 
the  rabbit.  The  fully  virulent  tissue  is  first  emulsified  with  sterile 
physiologic  salt  solution  in  the  proportion  of  1  to  100.  Then  the 
following  dilutions  are  prepared:  1  to  200,  1  to  500,  1  to  1000,  1  to 
2000,  1  to  5000,  1  to  10,000.  Three  c.c.  of  the  last-named  dilution 
forms  the  first  injection,  and  finally  1  c.c.  of  the  strongest  vaccine; 
i.  e.,  1  to  100  is  given  on  the  twentieth  day  of  the  treatment.  Babes 
uses  a  fixed  virus  which  has  been  attenuated  by  being  heated  to  80°  C. 
Tizzoni  and  Catanni  use  a  virus  attenuated  by  artificial  gastric  juice. 

There  have  now  been  treated  by  Pasteur's  original  method  or  one 
of  its  modifications  many  thousand  persons  bitten  by  rabid  animals, 
and  the  mortality  among  those  treated  is  less  than  1  per  cent.  This 
result  shows  clearly  the  great  efficiency  of  the  method.  It  is  believed 
that  the  results  will  still  be  improved  by  using  for  the  vaccination  less 
attenuated  and  more  virulent  fixed  virus,  because  it  appears  from  the 
work  of  Marx,  Babes,  and  Nitch  that  the  fixed  virus  of  the  rabbit 
for  man  in  subcutaneous  injection  represents  already  an  attenuated 
virus.  Kraus  believes  that  the  best  method  of  inoculation  after  bites 
of  rabid  animals,  particularly  after  extensive  lacerations,  will  be  the 
subcutaneous  injection  of  comparatively  large  doses  of  the  virulent 
fixed  virus. 

Cord  Lesions  after  the  Pasteur  Treatment. — The  Pasteur  treatment 
for  rabies  does  not,  in  the  vast  majority  of  cases,  lead  to  any  disturb- 
ances due  to  the  injections  themselves.  However,  there  have  been 
reported  a  number  of  cases — less  than  1  for  each  1000  cases  treated — 
in  which  the  injections  appear  to  have  led  to  some  transitory  lesions 
in  the  cords  of  the  persons  treated.  These  lesions  manifest  themselves 
by  disturbances  of  sensation,  disturbance  of  the  reflexes,  slight  paresis 
or  paralysis  and  the  symptoms  developed  sometimes  resemble  some- 
what the  very  serious  disease  known  as  Landry's  paralysis.  That 
these  symptoms  are  not  an  abortive  form  of  rabies,  but  due  in  some 


616  RABIES  AND  THE  NEGRI  BODIES 

way  to  the  injection  of  the  fixed  virus,  is  shown  by  the  fact  that  they 
have  occurred  in  persons  who,  as  shown  subsequently,  were  not 
bitten  by  rabid  dogs,  but  who  received  the  Pasteur  treatment.  Almost 
all  of  these  cases  end  in  complete  recovery.  There  is,  however,  one 
case  reported,  that  of  a  man  sixty-two  years  old,  who  died,  but  it  is 
doubtful  whether  he  died  in  consequence  of  the  Pasteur  treatment 
or  from  some  other  cause. 

Differences  in  Virulency  between  Street  and  Fixed  Viruses. — The 
fixed  virus,  while  of  a  maximum  virulency  subdurally  for  rabbits 
and  other  animals,  if  injected  by  any  other  route,  exhibits  a  de- 
creased virulency  and  can  be  used  for  immunizing  purposes.  Marx, 
discussing  the  differences  between  street  and  fixed  virus,  comes 
to  the  following  conclusions:  The  fixed  virus  produces  a  more 
abundant  or  a  more  powerful  toxin  than  the  street  virus.  The  rate 
of  velocity  of  multiplication  of  the  fixed  virus  is  greater  than  that 
of  the  street  virus;  the  fixed  virus  in  purely  subcutaneous  inoculation 
is  absolutely  harmless  for  man  and  apparently  much  less  virulent  for 
animals  than  the  street  virus.  For  some  animals  it  is  also  less  virulent 
in  intramuscular  (monkey)  and  in  intraocular  (monkey,  rabbit)  inoc- 
ulation. This  behavior  can  only  be  explained — everything  being 
equal — by  assuming  that  the  fixed  virus  is  more  easily  destroyed  by 
the  protective  powers  of  the  body  than  the  street  virus. 

The  question  then  naturally  arises,  Why  should  the  stronger,  more 
rapidly  multiplying,  fixed  virus  be  more  easily  destroyed  by  the 
protective  powers  of  the  body?  Marx  and  others,  discussing  this 
point,  do  not  offer  any  explanation.  The  following,  however,  suggests 
itself  to  the  author: 

In  natural  infection  the  virus,  in  order  to  produce  rabies,  must  be 
able  to  wander  in  some  way  from  the  portal  of  entrance,  mainly 
along  the  peripheral  nerves  to  the  central  nervous  system,  there  to 
invade  the  ganglion  cells,  to  multiply  and  to  produce  the  specific 
disease  hydrophobia.  By  injecting  the  street  virus  for  a  number  of 
generations  subdurally,  a  method  of  procedure  which,  of  course,  is 
highly  unnatural,  we  artificially  breed  an  abnormal  race  of  micro- 
organisms. These  become  more  highly  specialized  in  their  parasit- 
ism and  lose  their  organs  of  locomotion  or  other  apparatus,  which 
enabled  them  to  travel  from  a  portal  of  entrance  in  the  subcutaneous 
connective  tissue  to  the  central  nervous  system.  The  statement 
about  the  loss  of  organs  of  locomotion  is,  of  course,  not  to  be  taken 
literally,  but  simply  in  the  same  sense  as  Ehrlich  has  depicted  as 
definite  geometrical  bodies,  antigens  and  antibodies,  amboceptors, 
complements,  etc.,  in  order  to  give  a  clear  idea  of  their  combination 
neutralization,  fixation,  lysis,  agglutination,  etc. 

If  the  microorganisms  of  rabies  in  consequence  of  continued  sub- 
dural  injection  have  lost  the  power  to  travel  from  any  outside  place 
toward  the  central  nervous  system,  they  will  be  destroyed  in  loco 
by  the  protective  powers  of  the  invaded  organism,  and  while  this 


ANTIRABIC  SERUM  617 

occurs  antibodies  will  be  formed  which  prevent  the  organisms  of  the 
street  virus  from  producing  an  attack  of  hydrophobia.  That  there 
is,  indeed,  a  morphologic  difference  between  the  street  virus  and 
the  fixed  virus  is  shown  by  the  fact  that  the  latter  never  produces  the 
large  Negri  bodies  but  only  the  smallest  forms  of  them. 

Resistance. — The  rabies  virus  possesses  a  moderate  resistance. 
However,  it  must  not  be  forgotten  that  the  action  of  antiseptics, 
etc.,  cannot  be  studied  upon  the  pure  virus,  as  it  is  always  more 
or  less  protected  by  the  natural  substances  or  body  fluids  in  which 
it  is  contained.  In  a  tabulation  by  Heim,  compiled  from  the  work 
of  different  authors,  the  following  figures  are  given:  The  rabies  virus 
is  destroyed  by  1  to  1000  bichloride  of  mercury  in  two  to  three  hours; 
1  per  cent,  carbolic  acid  in  two  to  three  hours;  5  per  cent,  carbolic 
acid,  5  per  cent,  salicylic  acid,  10  per  cent,  sulphate  of  copper,  1  per 
cent,  kreolin  in  five  minutes;  70  per  cent  alcohol  in  twenty-four 
hours.  Formalin  vapors  fifteen  to  forty-five  minutes;  gastric  juice 
after  twenty-four  hours;  exposure  to  45°  C.  in  twenty-four  hours; 
50°  C.  in  one  hour;  52°  to  58°  C.  one-half  hour;-  60°  C.  very  rapidly. 
Low  temperatures  have  no  effect  upon  the  virus.  Frothingham  found 
the  cord  of  a  rabid  dog  kept  at  — 40°  C.  virulent  after  one  year  and 
ten  months.  The  virus  remains  virulent  longer  in  buried  cadavers 
than  in  those  exposed  to  the  air  and  light.  Glycerin  does  not  destroy 
the  virus,  in  fact,  it  acts  as  a  conservative  for  it  for  months. 

Antirabic  Serum. — Babes  and  Lepp  were  the  first  to  show,  as  early 
as  1889,  that  the  serum  of  dogs  immunized  by  Pasteur's  method 
contained  substances  which  are  antagonistic  to  rabies  virus,  and, 
therefore,  can  be  used  to  neutralize  it,  and  also,  as  is  claimed,  be 
employed  to  protect  dogs  by  passive  immunization  against  inoculation 
with  fixed  virus  and  against  the  bites  of  dogs  suffering  from  hydro- 
phobia. These  early  experiments  of  Babes  and  Lepp  appear  to  have 
been  the  first  to  demonstrate  the  formation  of  antibodies  against  a 
virus,  and  of  the  employment  of  such  antitoxin  to  produce  what  is 
now  universally  known  as  passive  immunization.  However,  the 
claim  that  antirabic  serum  can  cure  hydrophobia  after  it  has  made 
its  appearance  or  can  favorably  modify  its  course  is  not  admitted  as 
correct  by  Kraus.  Babes  and  those  who  have  used  an  antirabic 
serum  on  man  bitten  by  rabid  animals  have  never  used  it  alone  but 
always  in  combination  with  fixed  virus.  In  other  words,  they  have 
used  the  simultaneous  method,  never  the  method  of  pure  passive 
immunization,  and  it  is  claimed  that  the  results  of  the  simultaneous 
method  have  not  been  better  than  the  uncombined  treatment  with 
fixed  virus.  The  antirabic  serum  of  Babes  is  prepared  by  first  treat- 
ing rabbits,  donkeys,  dogs,  and  sheep  by  Pasteur's  method  and  sub- 
sequently injecting  increasing,  finally  large,  doses  of  fixed  virus.  The 
blood  of  the  hyperimmunized  animals  is  drawn  ten  to  twenty-five 
days  after  the  last  injection  of  virus  and  the  blood  serum  is  obtained 
in  the  same  manner  as  in  the  case  of  diphtheria  or  tetanus  antitoxin 


618  RABIES  AND  THE  NEGRI  BODIES 

preparation.  The  immune  serum  is  standardized,  according  to  Kraus, 
with  a  fixed  virus  prepared  fresh  from  the  medulla  of  a  rabbit  in 
the  proportion  of  1  part  emulsified  with  100  parts  of  physiologic  salt 
solution  and  filtered  through  a  paper  filter  to  remove  the  remaining 
coarse  particles.  Antiserum  and  virus  are  mixed  in  varying  propor- 
tions and  are  allowed  to  stand  for  twenty-four  hours  at  room  tem- 
perature. The  mixtures  are  then  injected  subdurally  into  various 
animals.  While  Kraus,  like  Babes  and  others,  succeeded  in  destroying 
the  fixed  virus  by  mixing  it  with  antiserum,  he  never  succeeded  in 
obtaining  any  protective  influence  by  injecting  the  antiserum  even 
in  large  doses  into  animals  which  were  subsequently  infected  with 
rabies. 

QUESTIONS. 

1.  What  are  the  other  scientific  and  common  names  for  rabies? 

2.  How  long  has  the  disease  been  known?     Where  does  it  occur? 

3.  How  is  it  generally  spread  in  natural  infection? 

4.  When  does  the  saliva  of  a  dog  suffering  from  rabies  become  infective  ? 

5.  What  animals  are  susceptible  to  rabies? 

6.  Discuss  the  period  of  incubation  of  rabies.     May  it  be  prolonged  to  one 
orjnore  years? 

7.  What  explanation  suggests  itself  as  to  such  a  long  period  of  incubation? 

8.  What  percentage  of  people  bitten  by  rabid  dogs  develop  rabies?    What 
percentage  of  dogs  bitten  by  other  dogs? 

9.  What  is  probably  the  behavior  of  the  rabies  virus  after  its  entrance  into 
the  body  of  persons  and  animals? 

10.  What  are  the  different  forms  of  rabies? 

1 11.  Describe  the  symptoms  of  furious  rabies  in  a  dog.    Also  of  dumb  rabies. 
*•  12.  What  is  the  consumptive  form  of  rabies? 

13.  Does  recovery  from  rabies  in  dogs  occur? 
*  14.  Describe  the  symptoms  of  rabies  in  man. 

15.  Describe  the  postmortem  findings  on  a  dog  dead  from  rabies. 

16.  Describe  the  changes  of  Van  Gehuchten  and  Nelis  in  the  cervical  and  spinal 
ganglia  in  rabies. 

17.  What  are  the  Negri  bodies?    In  what  parts  of  the  central  nervous  system 
would  you  look  for  them? 

18.  Describe  their  morphology  in  the  brain  of  a  dog  dead  from  rabies. 

19.  What  is  the  meaning  of  street  virus  and  of  fixed  virus? 

20.  What  is  the  difference  between  the  Negri  bodies  in  street  and  in  fixed 
virus  cases? 

21.  Are  the  Negri  bodies  found  in  the  earliest  stages  of  rabies?    If  not,  what 
practical  measures  should  be  adopted  in  the  diagnosis  of  rabies  by  the  aid  of  the 
Negri  bodies? 

22.  Give  in  detail  the  steps  in  the  rapid  microscopic  diagnosis  of  rabies  in  the 
dog. 

23.  Describe  the  best  fixing  and  staining  methods  used  in  this  rapid  diagnosis. 

24.  How  should  material  from  a  suspected  rabid  dog  be  prepared  for  sub- 
sequent laboratory  examinations  ? 

25.  What  are  the  opinions  as  to  the  nature  of  the  Negri  bodies? 

26.  What  are  the  peculiar  biologic  properties  of  the  Negri  bodies  which  enable 
them  to  survive  as  a  race  of  intracellular  parasites? 

27.  How  does  the  virus  of  rabies  spread  in  the  body  of  susceptible  animals 
from  its  portal  of  entrance  to  the  central  nervous  system? 

28.  Describe  the  method  to  inoculate  rabbits  subdurally  with  the  street  virus 

29.  How  is  the  fixed  virus  prepared  from  the  street  virus? 

30.  How  long  is  the  period  of  incubation  of  the  fixed  virus  inoculated  sub- 
durally? 

31.  Is  the  fixed  virus  in  subdural  inoculation  virulent  for  animals  other  than 
rabbits? 


QUESTIONS  619 

32.  What  is  the  principle  of  the  Pasteur  treatment  of  rabies? 

33.  How  is  the  material  for  this  treatment  obtained,  prepared,  and  used  on 
persons  ? 

34.  What  forms  of  rabies  is  seen  in  rabbits? 

35.  What  is  Babes'  fluid  ?    For  what  purpose  used  ? 

36.  In  what  kinds  of  injuries  is  the  most  energetic  Pasteur  treatment  to  be 
used? 

37.  What  other  methods  are  used  to  attenuate  the  fixed  virus? 

38.  What  would  be  the  effect  of  injecting  a  large  dose  of  fixed  virus  sub- 
cutaneously  into  a  person  ? 

39.  Does  the  Pasteur  treatment  sometimes  lead  to  any  disturbance  due  to  the 
inoculations?    If  so,  what  kind  of  disturbances? 

40.  Discuss  the  differences  in  virulency  between  the  street  and  the  fixed 
viruses  in  subcutaneous  inoculation. 

41.  What  hypothesis  have  you  to  offer  to  explain  these  differences? 

42.  Discuss  the  resistance  of  the  virus  of  rabies. 

43.  What  is  known  about  the  preparation  and  effect  of  an  antirabic  (immune) 
serum? 

44.  What  effective  method  is  there  to  protect  an  animal  by  passive  immuni- 
zation against  rabies?    What  is  the  curative  value  of  the  rabies  immune  serum? 


APPENDIX. 


Metric  System. — The  metric  system,  which  is  now  universally 
employed  in  all  scientific  measurements,  and  also  in  every-day  life  in 
most  European  countries,  is  based  upon  the  meter  as  the  primary 
object  of  length.  One  meter  is  equal  to  i  o  o  0*0  o  o  o  Par*  °f  the  dis- 
tance  measured  on  a  meridian  of  the  earth  from  the  equator  to  the 
pole  and  equals  about  39.37  inches.  The  original  meter  is  a  platinum 
bar  kept  in  the  public  archives  of  France  and  from  this  original 
standard  other  nations  have  procured  copies.  The  metric  system, 
based  upon  the  meter,  is  a  decimal  system,  i.  e.,  one  in  which  all 
values  are  fractions  in  tenths  or  multiples  of  ten.  The  meter  is  first 
divided  into  100  equal  parts  and  T^Q  meter  is  called  a  centimeter; 
each  centimeter  is  subdivided  into  10  parts  and  this  length  is  called 
a  millimeter.  For  microscopic  measurement  the  millimeter  is  again 
subdivided  into  1000  parts,  and  this  unit  is  called  1  micromillimeter, 
one  micron,  or  I/*.  The  following  are  the  most  important  sub- 
divisions : 


1  micromillimeter 

= 

0.001  millimeter 

1  millimeter 

= 

0.001  meter 

1  centimeter 

= 

0.01    meter 

1  decimeter 

= 

0.1      meter 

1  meter 

1  decameter 

= 

10          meters 

1  hectometer 

= 

100         meters 

1  kilometer 

= 

1000          meters 

1  myriameter 

= 

10,000         meters 

EQUIVALENTS  IN  INCHES. 

1  micromillimeter  =  0 . 0000394  inch 

1  millimeter  =  0 . 0394        inch 

1  centimeter  =  0.3937        inch 

1  meter  =  39.37  inches 

The  surface  measures  derived  from  the  meter  are: 

1  square  centimeter 
1  square  meter 

100  square  meters  =   1  Are         =     119.6      square  yards 
10,000  square  meters  =  1  Hectare  =         2.471  acres 

The  cubic  measures  are: 

1  cubic  centimeter     =  0 . 27     fluidrachm 
10  cubic  centimeters  =  0 . 338  fluidounce 
1000  cubic  centimeters  =   1  liter  =  0.909  quart 

One  cubic  centimeter  of  distilled  water  at  4°  C.  in  the  metric  system 
has  been  taken  as  the  unit  of  the  system  of  weight.  This  mass  of  water, 
equal  to  1  cubic  centimeter  (1  c.c.),  is  called  one  gram,  and  from  it 
the  following  weights  are  derived : 


622  APPENDIX 

1  milligram  =  0.001  gram  =  0.0154  grain  (avoirdupois). 

1  centigram  =  0.01    gram  =  0.1 543  grain 

1  decigram  =  0.1      gram.  =  1 . 5432  grain 

1  gram  =  15.432    grains 

1  kilogram  =  1000.         grams  =  2.2046  pounds. 

The  following  table  shows  the  equivalents  in  the  metric  system  to 
some  of  the  common  weights  and  measures : 

1  inch  2 . 54      centimeters 

1  foot  =         0.3048  meter 

1  yard  0.9244  meter 

1  rod  5.029    meters 

1  mile  1 . 0933  kilometers 

1  square  inch  6 . 452    square  centimeters 

1  square  foot  0 . 0929  square  meter 

1  square  yard  0.8361  square  meter 

1  acre  0.4047  hectare 

1  square  mile  =     259.  hectare 

1  cubic  inch  =       16 . 29      cubic  centimeters 

1  liquid  quart  0 . 9465  liter 

1  liquid  gallon  3 . 786    liters 

1  ounce  avoirdupois      =       28.35      grams 

1  pound  avoirdupois     =         0 . 4536  kilogram 

1  ounce  Troy  =       31 . 104    grams 

1  pound  Troy  0 . 3732  kilogram 

Thermometer  Scales. — The  principle  on  which  thermometers  are 
constructed  is  the  following:  A  fine  glass  tube,  having  a  closed  bulb 
on  its  lower  end,  contains  chemically  pure  mercury.  The  latter  is 
heated  in  a  boiling,  strong,  salt  solution.  The  mercury  then  rises  in 
the  tube,  evaporates  partly,  and  expels  all  the  air  from  the  upper 
open  end  of  the  tube.  If  now  this  upper  end  is  fused  and  the  mercury 
allowed  to  cool  a  vacuum  is  formed  above  it,  and  when  the  mercury 
again  expands  it  meets  with  no  resistance.  The  thermometer  tube, 
after  having  been  sealed  at  the  upper  end,  is  placed  in  melting  ice  and 
the  point  to  which  the  mercury  column  reaches  is  marked  the  freezing 
point  of  water  (zero  =  0).  The  thermometer  is  then  immersed  in  boil- 
ing distilled  water  and  the  point  to  which  the  expanded  mercury  now 
reaches  is  marked  as  the  boiling  point  of  water.  The  space  between 
the  freezing  point  of  water  and  its  boiling  point  is  marked  off  into 
a  number  of  equal  degrees.  In  the  Celsius  scale  the  space  is  marked 
off  into  100  equal  parts,  but  in  the  Reaumur  scale  into  80  equal  parts. 
The  thermometer  scale  most  commonly  in  use  in  this  country  in  every- 
day life  originated  in  a  somewhat  ridiculous  manner.  Fahrenheit, 
living  in  northeastern  Prussia,  on  a  very  cold  winter  day,  adopted 
the  then  prevailing  temperature  as  the  zero  point  of  his  scale.  He 
divided  his  thermometer  into  212  equal  degrees  between  his  zero 
and  the  boiling  point  of  distilled  water.  The  freezing  point  of  water 
on  the  Fahrenheit  scale  is  situated  at  32°  F.  There  are,  therefore, 
the  following  rules  for  changing  one  of  the  three  scales  into  another: 

1.  n  degrees  Reaumur  =  n  X     £  degrees  Celsius 

2.  n  degrees  Celsius  =  n  X    *  degrees  Reaumur 

3.  n  degrees  Reaumur  =  n  X     £  +  32  degrees  Fahrenheit 

4.  n  degrees  Celsius  =  n  X    f  +  32  degrees  Fahrenheit 

5.  n  degrees  Fahrenheit  =  n  -^  32  X    |  degrees  Reaumur 

6.  n  degrees  Fahrenheit  =  n  -f-  32  X    f  degrees  Celsius 


THERMOMETER  SCALES 


623 


The 

following  table  gives 

the  comparative  values  in  the 

three 

systems  of  thermometers: 

Cels. 

Fahr. 

Rdau. 

Cels. 

Fahr. 

Reau. 

Cels. 

Fahr. 

Re-au. 

-40 

-40 

-32 

22 

71.6 

17.6 

84 

183.2 

67.2 

-39 

-38.2 

-31.2 

23 

73.4 

18.4 

85 

185.0 

68.0 

-38 

-36.4 

-30.4 

24 

75.2 

19.2 

86 

186.8 

68.8 

-37 

-34.6 

-29.6 

25 

77.0 

20.0 

87 

188.6 

69.6 

-36 

-32.8 

-28.8 

26 

78.8 

20.8 

88 

190.4 

70.4 

-35 

-31 

-28 

27 

80.6 

21.6 

89 

192.2 

71.2 

-34 

-29.2 

-27.2 

28 

82.4 

22.4 

90 

194.0 

72.0 

-33 

-27.4 

-26.4 

29 

84.2 

23.2 

91 

195.8 

72.8 

-32 

-25.6 

-25.6 

30 

86.0 

24.0 

92 

197.6 

73.6 

-31 

-23.8 

-24.8 

31 

87.8 

24.8 

93 

199.4 

74.4 

-30 

-22 

-24 

32 

89.6 

25.6 

94 

201.2 

75.2 

-29 

-20.2 

-23.2 

33 

91.4 

26.4 

95 

203.0 

76.0 

-28 

-18.4 

-22.4 

34 

93.2 

27.2 

96 

204.8 

76.8 

-27 

-16.6 

-21.6 

35 

95.0 

28.0 

97 

206.6 

77.6 

-26 

-14.8 

-20.8 

36 

96.8 

28.8 

98 

208.4 

78.4 

-25 

-13 

-20 

37 

98.6 

29.6 

99 

210.2 

79.2 

-24 

-11.2 

-19.2 

38 

100.4 

30.4 

100 

212.0 

80.0 

-23 

-  9.4 

-18.4 

39 

102.2 

31.2 

101 

213.8 

80.8 

-22 

-  7.6 

-17.6 

40 

104.0 

32.0 

102 

215.6 

81.6 

-21 

-  5.8 

-16.8 

41 

105.8 

32.8 

103 

217.4 

82.4 

-20 

-  4 

-16 

42 

107.6 

33.6 

104 

219.2 

83.2 

-19 

-  2.2 

-15.2 

43 

109.4 

34.4 

105 

221.0 

84.0 

-18 

-  0.4 

-14.4 

44 

111.2 

35.2 

106 

222.8 

84.8 

-17 

1.4 

-13.6 

45 

113.0 

36.0 

107 

224.6 

85.6 

-16 

3.2 

-12.8 

46 

114.8 

36.8 

108 

226.4 

86.4 

-15 

5.0 

-12.0 

47 

116.6 

37.6 

109 

228.2 

87.2 

-14 

6.8 

-11.2 

48 

118.4 

38.4 

110 

230.0 

88.0 

-13 

8.6 

-10.4 

49 

120.2 

39.2 

111 

231.8 

88.8 

-12 

10.4 

-   9.6 

50 

122.0 

40.0 

112 

233.6 

89.6 

-11 

12.2 

-   8.8 

51 

123.8 

40.8 

113 

235.4 

90.4 

-10 

14.0 

-  8.0 

52 

125.6 

41.6 

114 

237.2 

91.2 

-   9 

15.8 

-  7.2 

53 

127  A 

42.4 

115 

239.0 

92.0 

-  8 

17.6 

-   6.4 

54 

129.2 

43.2 

116 

240.8 

92.8 

-   7 

19.4 

-  5.6 

55 

131.0 

44.0 

117 

242.6 

93.6 

-   6 

21.2 

-  4.8       56 

132.8 

44.8 

118 

244.4 

94.4 

-   5 

23.0 

-  4.0 

57 

134.6 

45.6 

119 

246.2 

95.2 

-  4 

24.8 

-  3.2 

58 

136.4 

46.4 

120 

248.0 

96.0 

-  3 

26.6 

-  2.4 

59 

138.2 

47.2 

121 

249.8 

96.8 

-  2 

28.4  . 

-   1.6 

60 

140.0 

48.0 

122 

251.6 

97.6 

-   1 

30.2 

-  0.8 

61 

141.8 

48.8 

123 

253.4 

98.4 

0 

32.0 

0 

62 

143.6 

49.6 

124 

255.2 

99.2 

1 

33.8 

0.8 

63 

145.4 

50.4 

125 

257.0 

100.0 

2 

35.6 

1.6 

64 

147.2 

51.2 

126 

258.8 

100.8 

3 

37.4 

2.4 

65 

149.0 

52.0 

127 

260.6 

101.6 

4 

39.2 

3.2 

66 

150.8 

52.8 

128 

262.4 

102.4 

5 

41.0 

4.0 

67 

152.6 

53.6 

129 

264.2 

103.2 

6 

42.8 

4.8 

68 

154.4 

54.4 

130 

266.0 

104.0 

7 

44.6 

5.6 

69 

156.2 

55.2 

131 

267.8 

104.8 

8 

46.4 

6.4 

70 

158.0 

56.0 

132 

269.6 

105.6 

9 

48.2 

7.2 

71 

159.8 

56.8 

133 

271.4 

106.4 

10 

50.0 

8.0 

72 

161.6 

57.6 

134 

273.2 

107.2 

11 

51.8 

8.8 

73 

163.4 

58.4 

135 

275.0 

108.0 

12 

53.6 

9.6 

74 

165.2 

59.2 

136 

276.8 

108.8 

•  13 

55.4 

10.4 

75 

167.0 

60.0 

137 

278.6 

109.6 

14 

57.2 

11.2 

76 

168.8 

60.8 

138 

280.4 

110.4 

15 

59.0 

12.0 

77 

170.6 

61.6 

139 

282.2 

111.2 

16 

60.8 

12.8 

78 

172.4 

62.4 

140 

284.0 

112.0 

17 

62.6 

13.6 

79 

174.2 

63.2 

141 

285.8 

112.8 

18 

64.4 

14.4 

80 

176.0 

64.0 

142 

287.6 

113.6 

19 

66.2 

15.2 

81 

177.8 

64.8 

143 

289.4 

114.4 

20 

68.0 

16.0 

82 

179.6 

65.6 

21 

69.8 

16.8 

83 

181.4 

66.4 

INDEX  TO  AUTHORS 


ABBOTT,  386 

Adametz,  517 

Addison,  338 

Alt,  571 

Anderson,  382,  481 

Anthony  and  Herzog,  431 

Aoyama  and  Myamoto,  402 

Archibald,  67 

Aristoteles,  304,  597 

Arloing,  187,  362 

Arloing,  Cornevin,  and  Thomas,  257 

Arthus,  84 

Ashburn  and  Craig,  545 

Aufrecht,  326 

Axe,  Mortley,  603 


B 


BABES,  309,  315,  581,  595,  602,  603,  613 

615,  618 

Babes  and  Lepp,  617 
Bahr,  403,  598 
Bailliart,  396 

Bang,  49,  212,  227,  228,  318,  319,  320, 
•  356,  359,  374,  413,  488 
Bang  and  Stribolt,  318 
Baruchelli,  200 
Bassenge,  485 
Baumeister,  300 
Baumgarten,  326,  331,  372 
Bayle,  325 
Beckmann,  290 
Beebe,  374 

Behring,  von,  272,  329,  357 
Berestnew,  413 
Bergey,  470,  488 
Bevan  and  Hamburger,  308 
Beyerinck,  461,  462,  465,  475,  476 
Blue  and  Wherry,  233 
Bodkin-Rodella,  476 
Bellinger,  20,  220,  224,  254,  326,  405, 

417,  451,  487 
Bellinger  and  Coskor,  448 
Bongert,  368 
Bonhoff,  487 
Bonome,  595 
Bordet  and  Danysz,  595 
Bordet  and  Gengou,  78 
Borgeaut,  374 

40 


Borrell,  238,  390 

Bose,  310 

Bostroem,  412,  413 

Bourgelat,  440 

Bournay,  417 

Bowhill,  593 

Boxmeyer,  283 

Brauell,  242 

Brauer,  318 

Brieger,  273 

Brieger  and  Ehrlich,  263 
I  Brinkerhoff,  373 
1  Broden,  558 

Brown,  31,  467 

Bruce,  381,  560,  561 

Brumpt,  558 

Briining,  488 

Buchner,  154,  273,  386 

Burr,  494 

Burri,  435 

Busenius  and  Siegel,  487 

Busse,  433 

Butterfield,  402,  404 


CAGNIARD-LATOUR,  20 

Calkins,  529,  531,  534,  536,  553,  554, 

565,  569,  605 
Calmette,  238,  356 
Carlr  263,  266 
Carre",  283 
Carter,  390 
Casper,  447 
Celli,  221 

Chauveau,  254,  326 
Chester,  318 
Choromausky,  314 
Christopher,  566,  593,  594 
Clegg,  372 
Cohn,  F.,  19,  27 
Cohnheim,  326,  331 
Coit,  504 
Conn,  462,  472,  500,  513,  514,  515,  516, 

518 

Conn  and  Esten,  488,  495 
Cooper,  571 
Cornet,  328,  359 
Cornet  and  Meyer,  351 
Cornevin,  300 
Councilman  and  Lafleur,  545  •?• 


626 


INDEX  TO  AUTHORS 


Courmont,  602 

Craig,  534,  540,  545,  546,  547,  548,  575 

Crawley,  562 

Curtice,  571 

Cushman,  549 


DALRYMPLE,  590,  591 

Danysz,  294 

Davaine,  20,  242,  504,  590,  591 

Dawson,  416,  417 

Dean,  373 

Dean  and  Todd,  486 

DeHaan,  416 

DeHaan  and  Hoogkamer,  416 

Delafond  and  Renault,  221 

Delepine,  481 

Denecke,  386 

De  Noble,  453 

De  Schweinitz  and  Dorsett,  281,  283 

De  Schweinitz  and  Smith,  228 

Desgallieras,  325 

Dickerhoff ,  488 

Dinwiddie,  284 

Doane-Buckley,  490 

Dobell,  532 

Dock,  545 

Dodd,  389 

Doflein,  569,  585 

Domec,  413 

Dor,  366 

Donovan,  566 

Dorsett,  283,  350 

Dorsett,  Bolton,  and  McBryde,  278,  281 , 

283 

Drigalski  and  Conradi,  286 
Ducleaux,  389,  479,  516,  517 
Dupuy,  294 
Dutton,  562 
Dutton  and  Todd,  387,  588 


EASTES,  488 

Eber,  481 

Eberle  and  Lange,  290 

Eddington,  445 

Ehrenberg,  387 

Ehrlich,  73,  74,  75 

Emmerich,  289 

Emmerich  and  Mastbaum,  301 

Eppinger,  402 

Ercolani,  422 

Erxleben,  20 

Escherich,  290 

Escherich  and  Pfaundler,  290 

Esmarch,  von,  147,  180 

Esmarch  and  Kokubo,  186 

Evans,  20,  553 

Ever,  356 

Eyre,  382 


F 


FESER,  254,  263,  487 

Ficker,  180 

Finkler  and  Prior,  386 

Fischer,  453 

Fish,  417 

Flexner,  267,  402,  486 

Flexner  and  Joblin,  377 

Fliigge,  190,  290 

Fliigge  and  Ostermann,  483 

Foster  and  De  Man,  507 

Frank,  318,  464,  488 

Frankel,  182,  267,  273,  372 

Frankel  and  Neufeld,  372 

Frankel- Weichselbaum,  376 

Frankland,  179,  180 

Franque,  376 

Freudenreich,  von,  494,  517 

Freudenwald,  115 

Friedlander,  379 

Frosch,  446,  542 

Frothingham,  430,  604,  607,  617 

Fuchs,  324,  325 


GAFFKY,  220,  486,  487 

Gaffky  and  Paak,  452 

Galli-Valerio,  593 

Galtier,  597,  603,  612 

Gamalaia,  351,  383 

Gartner,  223,  451,  452 

Gayon  and  Dupetit,  461 

Gerlach,  326 

Gilchrist,  430 

Goodlad,  325 

Gosswinter,  598 

Gotschlich,  190 

Grabert,  369 

Grassberger,  477 

Grassberger  and  Schattenfroh,  260,  261 

Grassi,  565 

Gray,  562 

Graybill,  586 

Grotenfeld,  472 

Gruber,  289,  471 

Gruby,  418 

Gruner,  597 

Guillebeau,  488,  489,  571 

Gurth,  325 


HAFFKINE,  238 
Hammarsten,  516 
Hankin,  238 
Hansen,  400,  466,  467 
Hare,  235 
Harrington,  190 
Harris,  545 
Harrison,  495 


INDEX  TO  AUTHORS 


627 


Hart,  424,  600,  603 

Hartenstein,  417 

Hart  maim,  548 

Hartz,  405 

Hecker,  446 

Heim,  617 

Heinemann,  488 

Heinemann  and  Glenn,  498 

Hektoen,  62,  334,  430 

Hellman,  612 

Helmholtz,  439 

Henderson,  494 

Henle,  20 

Herbert,  481 

Hering,  325 

Hertwig,  536 

Hess,  481,  482 

Hess  and  Degroix,  571 

Hewlett  and  Barton,  487 

Heymans,  482 

Hippokrates,  269,  324,  325 

Hodenpyle,  351 

Hoegyes,  599,  615 

Holman,  311 

Home,  266 

Hottinger,  283 

Howard,  Jr.,  267 

Hunter,  233,  235 

Hiippe,  220,  225,  436,  472 

Hutcheson,  595 

Hutyra,  230,  251,  266,  283,  357 

Hutyra  and  Marek,  221,  356,  360,  376 

Hyde  and  Montgomery,  431 


ISRAEL,  405 


JAKSCH,  VON,  459 

Janssen,  17 

Jensen,  203,  204,  223,  227,  241,  263,  264, 

265,  266,  292,  300,  356,  488,  500,  509, 

517 

Jess,  447 
Joest,  228 

Johne,  243,  373,  376,  405,  413 
Johne  and  Frothingham,  374 
Jordan  and  Harris,  393,  395 
Jiirgens,  547 


KALT,  598 

Kanthack  and  Sladen,  481 
Karlinski,  203,  487 
Kartulis,  545,  579 
Kastle,  508 
Kaumanns,  593 


Kekule,  73 

Kemper,  456 

Kent,  565 

Kerr  and  Novy,  266 

Kerwin,  325 

Kingberg,  265 

Kirioshita,  593,  594 

Kirchner,  379,  599 

Kitasato,  20,  235,  248,  254,  269 

Kitt,  206,  220,  221,  223,  224,  254,  256, 
258,  263,  265,  266,  293,  300,  301,  305, 
326,  344,  418,  487,  488 

Klebs,  20,  326 

Klein,  367,  481,  487 

Klenke,  325 

Kniippel,  486 

Knutt,  482 

Koch,  Robert,  19,  20,  21,  27,  144,  150, 
175,  187,  242,  263,  302,  324,  326,  343, 
350,  361,  385,  387,  390,  444,  445,  505, 
545,  558,  562,  576,  579,  585,  595 

Koch  and  Schutz,  362 

Koch  and  Wolfhiigel,  186 

Koch,  Kolle,  and  Turner,  444 

Kolle,  238 

Kolle  and  Turner,  444 

Konradi,  485 

Korn,  372 

Koske  and  Hertel,  507 

Kossel,  483 

Kossel  and  Weber,  585 

Krai,  418 

Kraus,  613,  615,  617,  618 

Krikoff,  599 

Kruse,  290,  486 

Kuetzing,  20,  466 

Kutscher,  311,  316,  366,  368 


LACKNER-SANDOVAL,  401 

Laennec,  325 

Lafar,  399,  426,  457,  478 

Lamble,  545 

Langenbeck,  405 

Langhans,  326 

Large,  376 

Laveran,  21,  390,  558,  572,  595,  596 

Laveran  and  Mesnil,  556,  567 

Laveran  and  Vallee,  390 

Law,  318 

Lebert,  405 

Leblanc,  595 

Leclainche,  293,  301 

Leclainche  and  Vallee,  265,  283 

Le  Count,  431 

Le  Due,  593 

Lehnert,  318 

Leichmann,  471,  513 

Leichtenstern,  294 

Leipert,  598 

Leishman,  566 

Lepelletier,  325 


628 


INDEX  TO  AUTHORS 


Leube,  458 

Leuckart,  551,  571 

Levy  and  Jacobsthal,  485 

Lewis,  553 

Liautard,  376 

Lienan,  374 

Ligniere,  205,  220,  221,  229,  230,  447, 

598,  602 

Ligniere  and  Spitz,  414 
Lingelsheim,  von,  200 
Lister,  472 
Loeffler,  212,  218,  220,  226,  228,  293, 

298,  302,  308,  446,  447 
Loeffler  and  Menge,  437 
Loeffler  and  Schiitz,  304 
Loeffler,  Schiitz,  and  Schottelius,  299 
Loehnis,  472 
Loesch,  545 
Lorenz,  301,  303 
Lorin,  307 

Lucet,  203,  417,  488,  489 
Luepke,  303 
Luckhardt,  395 
Lux,  494 


MACCALLUM,  402,  576 

MacCallum  and  Buckley,  376 

MacConkey,  494 

MacNeal  and  Kerr,  320 

MacNeal,  Latzer,  and  Kerr,  267 

McBryde,  283 

McCarthy  and  Ravenel,  376 

McClintock,  Boxmeyer,  and  Siffer,  283, 

284 

McCoy,  232,  233 
McFadyean,  306,  314,  315,  355,  441, 

445,  486,  487,  571 
McFadyean  and  Stockman,  320 
McFarland,  352 
Maffucci,  357 
Malmsten,  418 
Marchand,  566 

Marchiafava  and  Bignani,  575 
Marchiafava  and  Celli,  572 
Marchoux,  251 

Marchoux  and  Salimbeni,  389 
Marek,  257 
Markus,  374 
Marpmann,  472 
Martin,  390,  417,  442,  536 
Marx,  612,  615,  616,  617 
Maupas,  536 
Melvin,  343,  356 
Melvin  and  Mohler,  422 
Mesnil,  536 

Metchnikoff,  56,  57,  61,  501 
Mieckley,  598 
Miquel,  290,  459 
Miyajima,  563 
Mohler  and  Rosenau,  446 
Mohler  and  Washburn,  213,  328,  361, 

364,  381 


Mollereau,  488 

Montgomery  and  Hyde,  431 

Monti,  292 

Moore,  218,  226,  227,  228,  254,  292,  318, 

376 

Moore  and  Smith,  228 
Morgagni,  325 
Morse,  296 

Musgrave  and  Clegg,  541,  546,  561 
Musser,  545 


N 


NAEGELI,  26 

Neal,  416 

Needham,  18 

Neelsen  and  Johne,  452 

Negri,  604 

Nicolaier,  269 

Nicolle,  595 

Nicolle  and  Comte,  390 

Nielsen,  265,  266 

Niles  and  Bolton,  283 

Nitch,  615 

Noc,  549 

Nocard,  203,  293,  294,  311,  315,  316, 
318,  355,  356,  364,  367,  370,  402,  403, 
440,  441,  442,  487,  488,  559,  571,  598 

Nocard  and  Leclainche,  229,  339,  344 

Nocard  and  Rosignol,  355 

Nocard  and  Roux,  439,  440,  612 

Noergaard,  258,  422 

Noergaard  and  Mohler,  205,  223,  369. 
370 

Norris,  390 

Norris  and  Larkin,  402 

Novy,  387,  556,  562,  566,  577 

Novy  and  Knapp,  387,  390,  556 

Novy  and  McNeal,  556,  563 

Novy,  McNeal,  and  Hare,  556 

Nowak,  320 

Nuttall,  267 

Nuttall  and  Graham,  593 

Nuttall  and  Smith,  594 


OBERMEIER,  387 

Obermiiller,  481 

Olt,  300 

Osier,  545 

Ostertag,  49,  212,  228,  321,  322,  377 

Ostertag  and  Hecker,  49,  322 

Ostertag  and  Stadies,  283 

Otto,  238 


PAECHINGER,  571 
Pajal,  290 
Pallin,  595 


INDEX  TO  AUTHORS 


629 


Paltauf,  598,  599,  600 

Park,  495 

Pasteur,  19,  20,  21,  160,  220,  221,  223, 
250,  263,  278,  400,  443,  466,  472,  475, 
505,  577,  597,  602,  611,  612 

Pasteur  and  Thuillier,  298 

Pasteur,  Chamberland,  and  Roux,  612 

Pansini,  187 

Pearson,  374,  446 

Peck,  417 

Perroncito,  221 

Persoon,  466 

Peters,  424 

Petri,  179,  300,  481 

Petruschky,  401,  402,  453 

Pfeiffer,  366,  367,  379 

Pfeiffer,  L.,  556 

Piana,  401,  417,  593 

Pilavios,  315 

Pina,  292 

Pirquet,  von,  357 

Plenicz,  20 

Pollander,  20,  242 

Pourcelot,  441 

Prazmowski,  462,  463,  475 

Preisz,  301,  302,  311,  318,  366,  368 

Preisz  and  Guinard,  369 

Pretter,  301 

Prowazek,  565,  566 

Prudden,  357 


RABE,  205,  402 

Rabinowitsch,  373 

Raebinger,  228,  443 

Ralliet  and  Lucet,  571 

Ravenel,  362,  417 

Ravenel  and  Smith,  352 

Recklinghausen,  von,  20 

Reimarus,  20 

Remlinger,  602 

Remlinger  and  Riffat-Bey,  610 

Renk,  470 

Rettger  and  Harvey,  295 

Reynolds,  225 

Ricketts,  431 

Rindfleisch,  von,  20 

Ritter,  294 

Rivolta,  204,  220,  221,  405,  417,  428, 

488,  570 
Robinson,  190 
Rodella,  517 
Rodgers,  508,  566,  567 
Rodgers  and  Ayers,  472,  499 
Roeckle,  417 
Roell,  598 

Rosenau,  187,  294,  495,  499,  506,  509 
Rosenau  and  Anderson,  84 
Rosenau  and  McCoy,  494 
Rosenbach,  269 
Rosenberger,  352 
Rosenow,  378 


Ross  and  Milne,  387 

Roth,  484 

Rothschild,  233 

Rouget,  558 

Roux,  180,  181,  254,  311,  602 

Roux,  Chamberland,  and  Thuilliers,  597 
\  Ruge,  576 
!  Russel,  494 
i  Russel  and  Hoffmann,  490,  492 

Rutherford,  Higgins,  and  Watson,  559 


S 


SABOURAUD,  418 

Sakharoff,  389 

Salm,  597 

Salmon,  221,  223,  227 

Salmon  and  Smith,  278 

Salmonsen,  326 

Sand,  204,  263      • 

Sanfelice,  433 

Schaffer,  603 

Schattenfroh  and  Grassberger,  475 

Schaudinn,  383,  390,  534,  536,  545,  546, 

547,    548,   563,    566,  569,    572,  574, 

575,  577 

Schaudinn  and  Hoffmann,  387 
Schilling,  558,  593 
Schneider  and  Bufford,  559 
Schottelius,  301 
Schroder  and  Dusch,  19 
Schroeder,  481,  482,  590 
Schroeder,  Colton,  and  Mohler,  352 
Schubert,  301 
Schiider,  485 
Schiiller,  326 
Schulz,  L.,  494 
Schulze,  Franz,  19 
Schumann,  290 

Schiitz,  203,  204,  205,  226,  301,  417 
Schwan,  Theodor,  19,  20 
Sclavo,  251 

Sedgwick  and  Batchelder,  494 
Sedgwick  and  Tucker,  180 
Seiffert,  379 
Semmer,  220 
Semmer  and  Nencki,  444 
Sheldon,  424 
Shiga,  486 
Shoemaker,  485 

Siedamgrotzky  and  Schlegel,  376 
Silberschmidt,  403 
Silvius,  325 
Simond,  233 
Simpson,  233,  486 
Sivari,  268 
Smith,  Th.,  21,  84,  138,  185,  220,  227, 

350,  358,  361,  362,  549,  550 
Smith  and  Kilbourne,  581,  583,  584,  585, 

590 

Smith,  F.,  and  Steel,  416 
Sobernheim,  248,  251,  252 
Sorillon,  402 


630 


INDEX  TO  AUTHORS 


Spallanzani,  18 

Spencer,  599 

Steele,  21 

Stefansky,  373 

Stengel,  545 

Steinberg,  197,  378 

Stewart,  490 

Stewart  and  Kinsley,  341 

Stiles,  571,  579 

Stokes,  490 

Stoklassa,  463 

Stone  and  Sprague,  493 

Storch,  508,  513 

Strauss,  310,  357 

Strauss  and  Wurz,  364 

Streit  and  Harrison,  376 

Stribolt,  319 

Strong,  238,  428,  429,  486,  557 

Sucksdorf,  290 

Swain,  598 

Swithinbank  and  Newman,  499 

Szabo,  598 


TAPPEINER,  326 

Theiler,  283,  390,  444,  445,  558,  595 

Thoeni  and  Weigmann,  517 

Thomas,  258 

Tidswell,  373 

Tissier  and  Gashing,  477 

Tizzoni  and  Catanni,  615 

Tjaden,  506 

Tokishige,  428,  429 

Toussaint,  221 

Trask,  485,  486 

Trevisans,  220 

Trolldenier,  403 

Trommsdorf,  490 

Tulloch,  562 


UHLENHUT,  283,  446 


VALENTINE,  553 

Valle,  536 

Van  der  Eeckhout,  374 

Van  Ermengem,  452,  454,  455 

Van  Gehuchten  and  Nelis,  604 

Van  Helmont,  18 

Van  Leewenhoeck,  18 

Varo,  17 

Vaughan,  485 

Vedder,  545 

Veratti,  292 

Vestea  and  Zagari,  598 


Viereck,  548 
Villemin,  325,  326 
Virchow,  325 
Voges,  301,  558 


WALDEYER,  20 
Walker,  540,  545,  546,  548 
Ward,  222,  473,  486,  491,  493,  504 
Wassermann,  78,  201,  228,  274,  283 
Wassermann,  Neisser,  and  Bruck,  79 
Weber,  362,  483 
Weber  and  Titze,  358 
i  Weigert,  334 

Weigmann,  472,  500,  506,  513,  517 
Weil,  372 
Weisenfeld,  290 
Welch,  267 
Werner,  549 

Wharthin  and  Olney,  402 

Wherry,  308 

Wherry  and  McCoy,  373 

White  and  McCampbell,  357 

Widal,  289 

Willems,  443 

Williams  and  Lowden,  605,  607 
i  Wilson  and  Brimhall,  225,  376,  377 

Winogradsky,  460,  464 

Winterbottom,  562 

Wladimiroff,  314 

Woertz,  376 

Wolf  and  Israel,  412 

Wolf-Eisner,  356 

Wooley,  436 

Wright,  567 

Wright  and  Douglass,  60 

Wronin,  461 

Wuthrich  and  Freudenreich,  von,  470 


XYLANDER  and  Bohtz,  283 


YERSIN,  235,  238 


Z 


ZEIT,  187,  308 
Ziemann,  390,  558,  595 
Zinke,  597 
Zopf,  26 

Zschokke,  403,  571 
Ziindel,  598 
Ziirn,  221 


GENERAL  INDEX 


ABERRATION  of  rays  of  light,  chromatic 

and  spherical,  89 

Abortion,  infectious,  in  cows,  318 
serum  tests  for,  320 
in  mares,  321 
Abscess,  cold,  336 
Acetic-acid  bacteria,  466 
Achorion  keratophagus,  422 

Schonleinii,  420 
Acid,  carbolic,  as  a  disinfectant,  188 

formation  of,  by  bacteria,  139 
Actinobacillosis,  414 
Actinomyces  bicolor,  403 

classification  and   morphology  of, 

408 

cultural  properties  of,  411 
gelatinous  degeneration  of,  411 
granule,  morphology  of,  410 
morphology    of    fungus    in    pure 

culture,  412 
resistance  of,  413 
staining  methods  for,  409 
Actinomycosis,  405 

natural  infection  with,  413 
pathological  changes  in,  405 
subcutaneous,  407 

Actinomycotic    material,    how    to    ex- 
amine microscopically,  408 
Aerobic  bacteria,  40 
Agar-agar,  culture  medium,  130 
filtration  and  sedimentation  of, 

131 
medium  for  cultivation  of  ameba, 

541 
Agglutination,  70 

test  for  glanders,  314 
Agglutinins,  69 
Aggressin,  59 
Air,  bacteriological  examination  of,  175 

exact  method  of,  176 
Albumin-free      solutions     as     culture 

media,  135 
Alcohol    conversion    into    acetic    acid, 

chemical  formula,  467 
as  a  disinfectant,  191 
formation  by  bacteria,  139 
Alcoholic    fermentation,    discovery    of 

its  cause,  20 

Allantiasis.    See  Botulismus. 
Altmann's  granules  in  protozoa,  530 


Amboceptor,  hemolytic,  77 

thermostabile,  77 

Ameba  coli,  545.    See  also  Entameba. 
in  hanging-plate  culture,  544 
how  raised  in  cultures,  541 
meleagridis,  549 
methods  of  staining,  540 
microscopic  study  of,  540 
morphology  of,  539 
pathogenic,  545 
Ammonia,  test  for,  141 
Amphitricha,  31 
Amylobacter,  475 
Anaerobic  bacteria,  40 
Analysis,  gravimetric,  520 

volumetric,  520 
Anaphylaxis,  84 

Animal  experiments  with  bacteria,  169 
Anisogamy,   535 
Anopheles  maculipennis,  573 
Antagonism  between  bacteria,  44 
Anthrax,  241 

bacillus  and  spores,  resistance  of, 

247 

asporogenous,  245 
cultural  properties  of,  245 
discovery  of,  242 
morphology  of,  242 
spore  formation  of,  243 
diagnosis  of,  248 
modes  of  infection  under  natural 

conditions,  248 
occurrence    and    pathogenesis   of, 

241 

pathological  lesions,  241 
preparation  of  suspected  material 

for  laboratory  diagnosis,  249 
prophylaxis  of,  250 
spores,  germination  of,  244 
symptomatic,  pathological  lesions, 

254 

vaccines  and  serumtherapy,  250 
Antibodies,  70 
Antigen,  71 

and  antibody,  union  of,  78 
Antiseptics,  186 
Antitoxic  units,  72 

value,  unexplained  loss  of,  85 
Antitoxins.     See  Toxins  and  antitoxins. 
Aphthae    epizooticse.       See    Foot-and- 
mouth  disease. 
Apiosoma.     See  Piroplasma. 


632 


GENERAL  INDEX 


Apparatus,  chemical,  necessary  in  work 

in  bacteriology,  520 
Archoplasm,  531 
Argas  miniatus,  as  transmitter  of  spiro- 

chete  infection,  389 
Aroma  microorganisms  in  preparation 

of  butter,  513 
Arthrospores,  36 

Ascomycetes,  spore  formation  of,  422 
Ascus  formation  in  blastomyces,  426 
Asepsis,  meaning  of  term,  45 
Aspergillus  as  cause  of   pneumonomy- 

cosis,  417 
Atricha,  31 
Attenuation,  55 
Autoclave,  125 
Autogamy,  533 
Auto-infection.  50 
Automyxis,  533 
Azotobacter  agilis,  465 

chroococcum,  465 


B 


BABESIA  bigemina,  581 

Bacilli,  acid-fast,  other  than    tubercle 

bacillus,  371 
of  hemorrhagic  septicemia  group, 

220 

shape  and  arrangement  of,  33 
Bacillus  acidi  lactici  of  Grotenfeld,  473 

of  Hueppe,  472,  473 
Aderholdi,  474 

aerogenes  capsulatus  (Welch),  267 
amylobacter  Gruber,  476 
anthracis,  241 

spread  through  milk,  487 
symptomatici.      See  '  Bacillus 

of  symptomatic  anthrax, 
anthracoides,  435 
avisepticus,  221  — 

animals  susceptible  to,  222 
immunization  against,  223 
pathological  lesions  due  to, 

221 

bipolaris  septicus,  221 
botulinus,  454 

toxin  production,  455 
bovisepticus,  224 
casei  of  Freudenberg,  474 
cholerae  suis,  277 

agglutination  of,  by  serum 
of  hyperimmunized 
hogs,  284 
morphology  and  cultural 

properties  of,  280 
coli  communis,  289,  475 

as  cause  of  white  scours 

in  calves,  292 
coagulation   of   milk   by, 

291 

indol  formation  by,  292 
occurrence  of,  290 


Bacillus  comma  of  Koch,  385 
crassus  pyogenes  bovis,  207 
cyanogenus,  436,  479 
Dellbriicki,  474 
denitrificans,  461 
diphtherise,  210 
avium,  218 

cultural  and  staining  proper- 
ties of,  211 

growth  of,  on  Loeffler's  blood- 
serum  mixture,  211 
in  milk,  486 
morphology  of,  210 
toxin  formation  of,  212 
distortus,  geniculatus  and  tennis. 

See  Tyrothrix. 
edematis    aerogenes    of    Sanfelice, 

267 

maligni  II  of  Novy,  267 
sporogenes  of  Klein,  267 
emphysematosus  (Frankel),  267 
enteritidis  of  Gartner,  451 

prophylactic  measures 

against,  453 
toxin  production  of,  45 
equisepticus,  228 
erysipelatis  suis,  298 
general  characteristics  of,  26 
lactimorbi,  393 

agglutination  tests  for,  395 
cultural  properties  of,   393 
lactis  acidi  Leichmann,  474 

aerogenes   of  Escherich,   473, 

475 

inocuus  of  Wilde,  473 
viscosus  of  Adametz,  473 
lactopropylbutyricus  of  Tissier  and 

Gashing,  477 
lactorubefaciens,  479 
leprse  in  man  and  rats,  372 
liquefaciens  pyogenes  bovis,  207 
mallei,     cultural     and     biological 

properties  of,  308 
morphology  of,  308 
resistance  of,  310 
megatherium,  435 
membranaceus  amethystinus,  479 
mesentericus  vulgatus,  478 

in  ripening  of  cheese,  516 
mycoides,  478 

roseus,  479 
necrophorus,  212 

cultural  and  biological  proper- 
ties of,  216 

diseases  caused  by,  212,  213 
morphology  and  staining  prop- 
erties of,  216 
of  Bang,  318 

of  chronic  dysentery  in  cattle,  373 
of  Conn  (No.  14),  473 
of  Danysz,  294 
of  Gastromycosis  ovis,  265 
of     hog     cholera.       See     Bacillus 
cholerse  suis. 


GENERAL  INDEX 


633 


Bacillus  of  infectious  abortion  in  cows 

(bacillus  of  Bang),  318 
of  Johne,  373 
of  Klebs-Loeffler,  211 
of  Kutscher,  316 
of  malignant  edema,  263 
properties  of,  264 
protective         inoculation 

against,  265 

of  Moeller,  371  J 

of  mouse  septicemia,  302 
of  Nicolaier.     See  Bacillus  tetani. 
of  Petri,'  371 
of  Preisz,'368 
of  psittacosis,  294 
of  Rabinowitsch,  372 
of  swine  erysipelas,  298 

morphology  and  cultural 

properties  of,  299 
of  symptomatic  anthrax,  254 
properties  of,  255 
resistance  of,  257 
toxins  and  antitoxins,  260 
vaccine  therapy,  257 
of  ulcerative  lymphangitis  in  horse, 

315    ' 

ovisepticus,  226 

pestis,  animals  susceptible  to.  233 
cultural  properties  of,  237 
morphology  and  staining  prop- 
erties of,  235 
occurrence  in  blood,  234 
of  bubonic  plague,  230 
pneunjtonise  of  Friedlander,  473 
prodigiosus,  436,  479 
proteins  vulgaris,  434 
Zenkeri,  435 
Zopfii,  435 

pseudo-anthracis,  435 
pseudotuberculosis  murium,  368 
ovis,  368 

morphology  of,  369 
properties  of,  cultural,  370 
rodentium,  366 
putrificus  of  Bienenstock,  477 
pyelonephritidis  bovis,  207 
pyocyaneus,  200 

cultural  amd  biological  proper- 
ties of,  201 

morphology  and  staining  prop- 
erties of,  200 
occurrence  and   pathogenesis 

of,  200 

pigment  formation  in,  201 
resistance  of,  202 
similarity  of  cultures  on  pota- 
toes to  glanders  bacillus,  309 
simulating    Strauss    test    for 

glanders,  200 
toxins,  201 
j     pyogenes  bovis,  207 

suis,  208 
V    radicicola,  461 

infectious  filaments  of,  463 


Bacillus      rhusiopathise      suis.        See 

Bacillus  of  Swine  erysipelas, 
rubefaciens,  436 
ruber  aquatilis,  436 
rubescens,  436 

saccharobutyricus  of  Klecki,  476 
sarcophysematos  bovis,  254 

of  whales,  266 
sputigenes  of  Pansini,  473 
subtilis,  478 

in  ripening  of  cheese,  517 
suisepticus,  226 

immunization  against,  228 
pathological  lesions  produced 

by,  227 
tetani,  269 

and  aerobic  bacteria,  270 
as  a  saprophyte,  271 
morphology  of,  269 
tuberculosis,  346 

Biedert's  sedimentation 
method  to  find  few  bacilli, 
348 

cultural  properties  of,  350 
destruction  in  milk  by  pasteur- 
ization, 507 
determination  of  presence  in 

milk,  483 
differences     between     human 

and  bovine  types,  361 
excreted  with  feces  of   cows, 

482 
how  to  obtain  pure  cultures 

of,  350 

to  stain  in  sections,  349 
stained,  347 
human  type,  reaction  curve  in 

culture,  362 
yirulency     toward 
cattle     and     other 
animals,  362 
in  milk,  481 
its    occurrence  in    circulating 

blood,  352 
morphology  of,  346 
of  avian  type,  364 
resistance  of,  351 
staining  properties  of,   346 
typhi  murium,  293 
typhosus,  285 

formation  of  agglutinins  in,  287 
in  spleen  of  cow,  485 
spread  by  milk,  484 
violaceus,  436,  479 

(var.  manila?),  436 
Bacteria,  acid-fast,  109 

aerobic  and  anaerobic,  40 
binary  division  or  fission  of,  32 
chromogenic,  42 
definition  of,  26 
denitrifying,  460 
Gram  negative,  108 

positive,  108 
identification  of,   164 


634 


GENERAL  INDEX 


Bacteria  in  tissues,  staining  of,  115 

multiplication  or  propagation  of, 
32 

nitrifying,  459 

non-pathogenic,  434 

number  of,  in  ripening  cheese,  518 

nutrition  of,  39 

of  lactic  acid,  classification  of,  472 

pleomorphous,  27 

position  among  organisms,  26 

pyogenic,    general    considerations, 

193 

in  cattle,  206 
in  domestic  animals,  203 

quantitative    estimation    in    milk, 
494 

size  of,  35 

structure  of  body  of,  30 
Bacterium  aceti,  466 

casei  of  Leichmann,  474 

caucasicum,  474 

cocciforme  of  Migula,  473 

Kuetzingianum,  466 

limbatum  of  Marpmann,  473 

Pasteurianum,  466 

phlegmasia  uberis,  293 

pullorum,  294 

radicicola.    See  Bacillus  radicicola. 

xynxanthum,  479 

Zopfii,  435 

Bacterines.     See  Vaccines. 
Bacterin-treatment,  63 
Bacteroids,  463 
Balantidium  coli,  550 
Balbiana  gigantea,  578 

Rileyi,  579 

Bang's  method  of  stamping  out  tuber- 
culosis among  cattle,  359 
Beer  wort,  134 
Berkefeld  filter,  160 
Black-head  in  turkeys,  549 
Black-leg  of  cattle,  254 

vaccine,  directions  for  use  of,  258 
Blastomyces  as  cause  of  tumors,  433 

general  characteristics  of,  426 

how  to  demonstrate  nucleus  of,  427 

of    dermatitis,    morphology,    and 
cultural  properties  of,  432 

spore  formation,  426 
Blastomycosis  farciminosus.     See  Lym- 
phangitis epizootic. 
Blepharoblast,  531 
Blood-agar,  133 

corpuscles,  red,  washed,  57 

examination     for    plasmodia     of 
malaria,  576 

serum  coagulator,  216 

sterile  as  a  culture  medium, 

how  obtained,  124 
sterilization  of,  125 
Bodies,  polar,  30 
Boophilus  bovis,  586 
Botryococcus  ascoformans,  205 
Botulismus,  454 


Botulismus  toxin,  455 
Bouillon,  nutrient,  exact  standardiza- 
tion of,  129 
how  prepared,  128 
Bovovaccine,  357 
Brahma  cattle  immunity  against  piro- 

plasma  infection,  593 
Breslau  regenerator  for  disinfection,  190 
Brownian  movement,  31 
Bubo  of  plague  in  rats,  232 
Buchner's  anaerobic  tube,  153 
Budding  fungi.    See  Blastomyces. 
Burner,  Bunsen's,  102 
Bursattee  in  horse,  416 
Butter  bacilli,  371 

bacteria  in,  515 

making,  bacteria  in,  513 
Butyric-acid  bacilli,  motile,  476 
non-motile,  476 

formers,  anaerobic,  475 


CADERAS  (mal  de  caderas),  558 
Camera  lucida,  97 
Canine  madness,     See  Rabies. 
Capsule  coccus  of  pneumonia,  378 
Capsules  of  bacteria,  staining  of,  109 

Friedlander's  method,  109 
Johne's  method,  109 
Ribbert's  method,  110 
Welch's  method,  110 
Caput  medusae  of  anthrax  cultures,  24 
Carbolic  acid,  test  for,  141 
Carbuncle.    See  Anthrax. 
Carmin  stain,  119 
Caseation  in  tuberculosis,  333 
Catarrh,  malignant,  of  cattle,  293 
Cattle  plague,  443 

immunization  against,  444 
diseases     transmissible     to     man 

through  milk,  487 
tick,  life  history  of,  586 
Celloidin,  embedding  method,  116 
j  Cells,  phagocytic,  57 
;  Centrosome,  533 
|  Cercomonas,  564 

1  Cerebrospinal  meningitis,  equine,  infec- 
tious, 376 

I  Chamber-land  filter,  160,  161 
Characteristics  of  cultures,  164 
Cheese  making,  bacteria  in,  513 

ripening    of,    by   microorganisms, 

516 

Cheeses,  rennet  milk  and  sour  milk,  516 
ripening  of,  by  microorganisms,  516 
Chemical  tests,  etc.,  used  in  bacterio- 
logical work,  520 

Chemicals,  effect  of,  upon  bacteria,  44 
Chemotaxis,  59 

positive,  negative,  and  indifferent, 

59 
Chicken-pox,  448 


GENERAL  INDEX 


635 


Chlamydospores,  400 

Chromatophores,  531 

Cilia,  protozoan,  532 

Cladothrices,  401 

Cladothrix,  401 

Clauss'  fan  bacillus,  473 

Clostridium  Americanum  of  Pringsheim, 

477 

butyricum,  475 
morphology  of,  36 
Pasteurianum,  464,  477 
sarcophysematos  bovis,  254 
Cocci,  classification  of,  33 

pathogenic,    for   domestic  animals 

and  man,  376 
Coccidia,  569 
Coccidiosis  in  cattle,  571 
in  sheep,  571 
renalis,  571 

Coccidium  cuniculi  (oviforme),  570 
fuscum,  571 
perforans,  571 
schubergi,  569 

tenellum  as  cause  of  white   diar- 
rhea of  chicks,  296,  571 
Coccus  cacti  cochinelifera,  523 

general  characteristics  of,  26 
Cochineal  indicator,  523 
Cohn's  solution,  136 
Cold,  effect  upon  bacteria,  187 
Collodion  sacs,  172 
Colonies,  fishing  for,  146 

how  to  count  them  on  plates,  174 
surface  details  and  peripheral  out- 
lines of,  169 

Colostrum  corpuscles,  489 
Colpitis  granulosa  infectiosa  bovum,  322 
Columella,  399 
Commensales,  24 

Complement,  deviation  or  fixation,  78 
hemolytic,  77 
thermolabile,  77 

Concave  slide  in  examination  of  bac- 
teria, 103 

Condenser,  Abbe"  or  substage,  88 
Conidia,  400 
Cover-glass    preparations    of    bacteria, 

how  to  stain  them,  105 
Cream,  bacteria  in,  515 
Culex  pipiens  as  carrier  of  proteosoma 

infection,  576 
Culture  media,   artificial,   transparent, 

123 
how  inoculated  in  bacteriologic 

water  examination,  181 
natural,  123 
sterilization  of,  123,  124 
medium  of  Drigalski  and  Conradi, 

286 
special,   for  Clostridium   pas- 

teurianum,  464 
for  trypanosomes,  556 
soil,  change  of  reaction  in,  43 
tubes,  how  labelled,  149 


Cultures/anaerobic,  150 

Buchner  pyrogallic-acid 

method,  154 
in  fluid  media,  151 
in  hydrogen  atmosphere,  151 
Koch's  original  method,  150 
how  obtained  from  bacteria  in  cir- 
culating blood,  147 
pure,  definition  of,  123 

how  obtained  from  pathologic 

material,  143 
in  study  of  bacteria,  87 
Curd,  ripening  of,  into  cheese,  517 
Cytosporon  danilewsky,  576 
Corynethrix    pseudo-tuberculosis    mu- 
irum,  368 


! "  DAUERFORMEN,"  35 
Death  point,  thermal,  of  bacteria,  39 
Decay,  definition  of,  458 
!  Dermatitis,  blastomycotic,  in  man,  430 
Dermatomycoses,  418 

methods  of  microscopic  examina- 
tion of,  418 

Diaphragm,  iris,  of  microscope,  88 
Diarrhea,  white,  of  chicks,  295 
Diastase,  fermentation  products  of,  138 

test  for,  138 
Digester,  125 

Dimethylamidoazobenzol  indicator,  523 
Diphtheria  antitoxin,  212 
Diphtheritic    inflammations,     bacteria 

productive  of,  210 
Diplococci,  definition  of,  33 
Diplococcus  intracellularis  equi,  377 
meningitidis,  377 

lanceolatus,  378 

pneumonias    of    Frankel-Weichsel- 

baum,  378 

Disease,  contagious,  definition  of,  53 
Disinfectants,  184 

chemical,  186 

effectiveness  of,  184 
Disinfection,  principles  of,  184 
Distemper  in  dogs,  447 
Division,  amitotic,  533 

mitotic,  533 
Doane  Buckley's  method  of  estimating 

leukocytes  in  milk,  490 
Dog  typhus,  bacillus  of,  230 
Dourine,  558 

Drying  out,  effect  of,  upon  bacteria,  187 
Dunham's  peptone  water,  135 
Dysentery,  amebic,  546 

bacilli  in  milk,  486 
Dysenteria  coccidiosa  bovum,  571 


ECTOPLASM  of  bacteria,  30 
protozoan,  530 


636 


GENERAL  INDEX 


Edema,    malignant,    natural    infection 

with,  264 

pathological  lesions  of,  263 
Egg-albumen  fixative,  how  prepared  and 

used,  118 

Electrical  currents,  effect  of,  upon  bac- 
teria, 44,  187 
Endogamy,  535 
Endospore,  definition  of,  35 
Endospores  of  hyphomycetes,  399 
Entameba  coli,  545 
hystolytica,  546 
tetragena,  548 
Enteritis,  hemorrhagic,  in  cows  causing 

infection  of  milk,  487 
Enterohepatitis  in  turkeys,  549 
Entoplasm  of  bacteria,  30 

protozoan,  530 
Enzymes  in  milk,  Storch's  test  for,  508 

secreted  by  bacteria,  42,  138 
Epidermophyton  gallinarum,  422 
Epithelioma  contagiosum  avium,  448 
Equine  disease,  local,  and  a  bacillus  of 

subtilis  group,  396 
Erlenmeyer  flask,  128 
Erysipelas  of  swine,  pathological  lesions 

of,  298 

Esmarch's  apparatus  for  counting  colo- 
nies, 175 
roll  tubes,  147 
Evaporation,     protection     of     culture 

media  against,  127 
Excretion  of  pathogenic  bacteria,  51 
Experiment  of  Schulze,  18 

of  Schwann,  18 
Exogamy,  535 
Exospores,  400 
Eyepieces,  microscopic,  89 
Eyre's  method  of  bacteriological  air  ex- 
amination, 177 


FARCY  in  cattle,  402 

Fehling's  standard  solution,  524 

Fermentation  by  bacteria,  42 
tube,  140 

Ferments,  proteolytic,  of  bacteria,  138 

Fever,  splenic.     See  Anthrax. 

Filters  for  bacterial  cultures,  160 

Filtrates,  germ-free  or  sterile,  162 

Fission  fungi,  26 

Flagella,  30 

protozoan,  various  types  of,  532 
staining  of,  111 

Bunge's  method,  112 
LoefHer's  method,  111 
Pitfield's  method,  112 
Van  Ermengem's  method,  112 

Foot-and-mouth  disease,  445 

protective  inoculation  against, 

446 
transmitted  through  milk,  487 


Foot-rot  of  sheep,  213 
Forceps  for  cover-glasses,  101 
Formaldehyde  as  disinfectant,  189 
Focus  of  objectives,  89 
Focussing  the  microscope,  90 
Fowl  cholera,  bacillus  of,  221 

plague,  447 
FrankeFs  soil  borer,  182 

solution,  136 

Frankland  and  Petri's  method  of  bac- 
teriological air  examination,  179 
Fusarium  equinum,  422 

moniliforme,  424 


;  GALACTOCOCCUS  albus,  489 
flavus,  489 
versicolor,  489 
Gametes,  535 

Gas  production  by  bacteria,  140 
G astro-enteritis   hemorrhagica   of   dog, 

230 

-intestinal  tract  as  a  portal  of  en- 
trance for  pathogenic  bacteria,  49 
Gelatin-agar,  133 

culture  medium,  130 
Gemmae  formation,  400 
Genital  tract  as  a  portal  of  entrance  for 

pathogenic  bacteria,  49 
j  Gentian  violet,  anilin-water,  107 
Giemsa's  stain,  109 
Giant  cells  in  tuberculosis,  332 
Glanders,  304 

agglutination  test  for,  314 
biological  test  for,  310 
in  man,  307 
mallein  test  for,  311 
mode  of  infection  in,  304 
pathological  lesions  of,  305 
pseudo-,  315 
pulmonary,  306 
Glassware,  101 

for  bacteriologic  work,  how  steril- 
ized, 126 
Glossina  longipennis,  561 

morsitans,  560 
Glucose-formate-gelatin,  132 
Glycerin  agar,  132 
Gonidia,  399 
Gonococcus,  380 
Gram's  decolorizing  fluid,  108 
method  of  staining,  107 
staining  method  for  paraffin  sec- 
tions, 120 
Gram-Weigert  staining  method  for  cel- 

loidin  sections,  121 

Granulations,  fungous,  tubercular,  335 
i  Granules,  sporogenous,  36 
Granulobacter  saccharobutyricus  Beye- 

rinck,  476 
Grass  bacillus,  371 

Growth  of  bacteria,  elements  necessary 
for,  40 


GENERAL  INDEX 


637 


HALTERIDIUM  infection  in  birds,  576 
Hanging  drop,   method   of  examining 

bacteria  in,  103 
Heat,  effect  of,  upon  bacteria,  44 

dry  and  moist,  effect  of,  upon  bac- 
teria, 186 

Hemameba  relicta,  576 
Hemoglobin-agar,  134 
Hemoglobinuria  in  sheep,  595 
of  cattle.     See  Texas  fever. 

in  Finland,  585 
Hemolysins,  77 

Hemolytic  system  or  chain,  77 
Hemophalis  leachii,  594 
Hemoproteus  infection  in  birds,  576 
Hemosporidia,  572 

in  birds,  576 
Hen's  eggs  as   medium   for  anaerobic 

cultures,  155  ^ 

Herpetomonas,  565 

donovani,  566,  567 
muscse  domesticae,  565 
Hesse's   method  of   bacteriological  air 

examination,  176 
Hog  cholera,  true  etiology  of,  281 

pathological  lesions  of,  278 
protective  inoculation  in,  283 
vaccine,  Bruschettini's,  285 
Horse  distemper,  228 

sickness,  African,  445 
Hyaloplasm,  protozoan,  530 
Hydrogen,    sulphuretted,    formed    by 

bacteria,  test  for,  140 
Hydrophobia.     See  Rabies. 
Hypersusceptibility,  84 
Hyphomycetes,  higher,  as  cause  of  dis- 
ease, 416 
lower,  398 
Hypotrichosis  localis  cystica,  571 


ICHTHYOSISMUS,  454 

Icterohematuria  in  sheep,  595 
Illuminator,  dark-field,  97 
Image,  microscopic,  89 
Immunity,  acquired,  72 

active  and  passive,  73 

congenital,  72 

definition  of,  58,  72 

natural  and  artificial,  58 

side-chain  theory  of  Ehrlich,  73 
Immunization,  simultaneous  method  of, 

73 

Immunizing  units,  definition  and  esti- 
mation of,  72 

Impression  preparation,  147 
Incubator,  156 

difficulty  in  regulating  of,  159 

how  started,  158 
Indicators,  522 


j  Indol,  test  for,  141 

!  Infection,  definition  of,  53 

of  cryptogenetic  origin,  50 
protective  agencies  in,  55 
protection    against,    by    phagocy- 
tosis, 57 

tuberculous,  cryptogenetic,  335 
Infectious  disease,  definition  of,  53 

early  theories  as  to  causes,  20 
Inflammation,    diphtheritic,    definition 

of,  210 

Influences  inimical  to  bacteria,  44 
Influenza,  equine,  228 

catarrhal  and  pectoral  form  of, 

229 

I  Infusoria,  537 

Inoculation,  exact  quantitative  deter- 
mination of,  171 
from  postmortem  material,  148 
subdural,  technique  of,  613 
Insects  as  carriers  of  pathogenic  micro- 
organisms, 50 

Intraperitoneal  inoculation,  170 
Intravenous  inoculation,  170 
Invertin,  demonstration  of,  139 
Involution  forms  of  bacteria,  29 
Isogamy,  535 


KALA-AZAR,  566 

Karyogonad,  531 

Karyokinesis,  533 

Karyomitosis,  533 

Kefir  granules,  480 

Kinoplasm,  531 

Kipp  apparatus  for  developing  hydro- 
gen, 152 

"  Klatsch-prseparat,"  147 

Klebs-Loeffler,  bacillus  of.  See  Bacillus 
diphtherias. 

Koch-Pfeil  safety  lamp,  157,  159 

Koch's  standpoint  on  intertransmissi- 
bility  of  bovine  and  human  tubercu- 
losis, 363 


L0,  L-f,  significance  of  symbols,  75 

"Lab"  enzyme,  test  for,  139 

Labferment.    See  Rennet. 

Lactic  acid,  bacteria,  471 

in  ripening  of  cheese,  517 
dextrogyr  and  sinistrogyr,  471 
how  formed  from  lactose,  471 

Lactose -litmus  agar,  133 
gelatin,  132 

Lamblia  intestinalis,  565 

Lamps,  101 
|  Leeches  in  horse,  416 

Leguminosse  and  nitrogen  fixation,  461 

Leishman-Donovan  bodies,  566,  567 

Leptothrices,  401 


638 


GENERAL  INDEX 


Leptothrix,  401 

Leucocidin,  196 

Leuconostoc  mesentericus,  474 

Leukocyte  count  in  milk,  variability  of, 

in  healthy  cows,  492 
Leukocytes  in  milk,  489 

methods  of  estimation  of,  490 
of  dog  and  phagocytosis  of  anthrax 

bacilli,  62 

serum  free,  and  lack  of  phagocy- 
tosis, 60 

how  prepared,  59 
Levaditti's  silvering  method  for  spiro- 

chete,  121 
Lime,  caustic,  as  a  disinfectant,  188 

chlorinated,  as  a  disinfectant,  188 
Lip-and-leg  disease  of  sheep,  213 
Litmus  gelatin,  132 
indicator,  523 
milk,  134 

Loeffler's  blood-serum  mixture,  133 
Lopophyton  gallinarum,  422 
Lopotricha,  31 

Lumpy  jaw.     See  Actinomycosis. 
Lung   plague   in   cattle.     See   Pleuro- 

pneumonia,  contagious. 
Lymphadenitis,  caseous,  of  sheep,  368 
pathological  lesions  in,  369 
Lymphangioitis      farciminosa      bo  vis, 

402 
Lymphangitis,  epizootic,  in  horses  due 

to  blastomyces,  428 
ulcerosa  pseudofarcinosa,  315 
Lysins,  69 
Lysis,  70 
Lyssa.    See  Rabies. 


M 


MACRONUCLEUS,  531 
Macrophages,  57 

MacFarland  apparatus  for  rapid  nitra- 
tion of  toxins,  161 
Malaria,  572 
Malarial  fever  curves,  572 

parasites,  572 

Mallein,  preparation  of,  311 
test,  311 

effect  of,  upon  horses,  312 

for  mules,  313 
Malleus,  304 
Mallory's    eosin-methylene-blue    stain, 

120 

Malta  fever,  381 
Manure,  green,  464 
Martin's  bouillon,  442 
Margaropus  annulatus,  586 
Mastigophora,  537 
Mastitis  in  cows,  bacteria  of,  487 
Maturation,  phenomena  of,  533 
Measure,  microscopic,  35 
Meat-poisoning,  bacteria  of,  451 
Membranelles  in  protozoa,  532 


Mercury  bichloride   as  a  disinfectant, 

188 

Merozoite,  535 

Metabolic  products  of  bacteria,  41 
Metachromatic  granules,  30 
Metric  system,  comparative  tables  of, 

621 

Methylene  blue,  Loeffler's  alkaline,  107 
Microbes,  22 

Micro-bunsen  burner,  159 
Micrococcus  agilis,  437 

butyri  aromafaciens,  475 
candicans  Fliigge,  475 
caprinus,  381 
catarrhalis,  379 
cirrhiformis  Migula,  479 
coronatus  Fliigge,  475 
lactis  acidi,  474 

Leichmann,  474 
Marpmann,  474 
viscpsi,  475 
melitensis,  381 
mucilaginosus,  474 
pyogenes  Rosenbach,  474 
tetragenus,  437 
urese,  458 

liquefaciens,  458 
Micrometers,    how    to    use    them    for 

measuring  bacteria,  95,  96 
Micro-gas  lamp,  157 
Micronucleus,  531 
Microorganisms,  22 

causing  fermentation,  23 
non-pathogenic,  23 
pathogenic,  22 

first  discovery  of,  20 
role  of,  in  nature,  23 
thermophile,  38 
thermotolerant,  39 
Microphages,  57 

Microscope,  first  construction  of,  18 
modern,  92 
parts  of,  88 

source  of  light  for  use  of,  87 
use  of,  in  study  of  bacteria,  87 
Microsomes  in  protozoa,  532 
Microsporidia,  569,  577 
Microsporon     audouini,     equinum     et 

caninum,  420 
Microtome,  119 

Milk,  acidity  of,  determination  of,  502 
alcoholic  fermentation  of,  479 
bacterial  analysis  of,  steps  in,  496 
counts,    interpretation   of   re 

suits  of,  499 
in  round  numbers,  502 
as  a  culture  medium  for  bacteria, 

134 

bacteria  in,  source  of,  470 
bacteriology  of,  469 
certified,  503 

chromogenic  bacteria  in,  479 
coagulation  of,  516 
Commission,  medical,  503 


GENERAL  INDEX 


639 


Milk,  condemnation    of,    on    basis    of 

bacterial  counts,  501 
diminution  of  bacteria  in,  494 
germicidal  property  of,  494 
lactic-acid  bacteria  in,  most  com- 
mon, 474 
laws  regulating  its  production  and 

sale  in  Germany,  501 
market,  tubercle  bacilli  in,  481 
method   of   estimating   leukocytes 

in,  490 

moulds  found  in,  480 
pasteurization  of,  504 

advantages  and  disadvantages 

of,  509 
at  home,  508 

changes  produced  by,   507 
objects  of,  506 
summary  of  objection  against, 

510 

pathogenic  bacteria  in,  481 
peptonizing  bacteria  in,  477 
sickness  of  cattle,  393 
tubercle  bacilli  in,  483 
typhoid  bacilli  in,  484 
Miescher's  tubules,  578 
Mixed  infection,  definition  of,  55 

virulent  types  of;  55 
Moist  chamber,  method  of  examining 

bacteria  in,  103 
Monotricha,  31 

Morbus   maculosus   equorum,    strepto- 
cocci in,  205 
Mucor  as  cause  of  pneumonomycosis, 

418 

Mucous    membrane    as    a    portal     of 

entrance  for  pathogenic  bacteria,  48 

Mycelium  of  moulds  or  hyphomycetes, 

398 

Mycoderma  aceti,  466 
Mycomycetes,  399 
Myonemes,  532 


N 


NAG  ANA,  558 
Negri  bodies,  604 

are  they  protozoa,  610 

in  fixed  virus,  606 

in  street  virus,  605 

methods  of  demonstrating  of, 
607 

morphology  of,  605 

stained  with  Giemsa's  solution, 

607 

eosin-methylene  blue,  608 
Neuroryctes  hydrophobias,  605 

peculiar  properties  of,  610 
Nicolaier,  bacillus  of.     See  Tetanus. 
Nitrate  water,  135 
Nitrification,  definition  of,  458 
Nitrites  formed  by  bacteria,  test  for,  141 
Nitrobacteria,  460 
Nitrogen  cycle,  bacteria  of,  457 


Nitrogen  fixation  of  free,  461 
Nitrogenous  compounds  in  milk,  516 
I  Nitrosococcus,  460 
I  Nitrosomonas  Africana,  460 

Europea,  460 

Japonica,  460 

Javanica,  460 
"Normaloese,"  172 
Normal  solutions,  how  prepared,  521 
Nosema  bombycis,  577 
Novy  jar  for  anaerobic  cultures,  153 
Nucleus  gonad,  531 


OBJECTIVES,  dry,  90 
microscopic,  89,  90 
oil-immersion,  90 

Objects,  stained  and  unstained,  how 
to  be  examined  with  microscope,  91, 
92,93 

Oidia  formation,  400 
Oidium  albicans,  424 

lactis,  400 

Ophthalmo-tuberculin  test,  356 
Opsonic  incubator,  61 
index,  62 

how    obtained,    with    horse's 

blood  serum,  63 
Opsonins,  60 

experimental  demonstration  of,  61 
Opsonizing  pipette,  61 
Organella  of  protozoa,  529 
Organisms,  22 

intracellular,  22 
Organs,  22 
I  Ookinet,  574 

Oxygen,  how  removed  from  air  for 
anaerobic  cultures,  154 


PARAFFIN  embedding  method,  117 

removal  of,  from  sections,  118 
Paraplectrum  fcetidum,  477,  517 
Parasites,  ectogenous  and  entogenous,  24 

facultative,  24 

strict  or  obligate,  24 
Paratyphoid  bacillus  in  milk,  485 
Pasteur  bulb,  19,  20 

culture  filter,  160 

flask,  128 

treatment  against  rabies,  612 

cord  lesions  after,  615 
Pasteurelloses,  220 
Pasteurelosis  avium,  221 
Pasteurization,  506 

definition  of  term,  505 
Pathogenic  bacteria,  occurrence  of,  in 

nature,  47 

(    precautions   in  working  with, 
106 


640 


GENERAL  INDEX 


Pearl  disease,  337 

Peptone  rosolic  acid  water,  135 

Periorchitis  in  guinea-pigs  produced  by 

bacillus  pyocyaneus,  200 
Peritricha,  31 
Pestis  equorum,  445 
gallinarum,  447 

Petri   dishes,    contamination    of,   with 
moulds,  146 

how  poured  for  bacteriologic 

examination,  144 
object  of  preparing,  145 
substitute  method  for,  146 
Phagocyte,  definition  of,  56 
Phagocytosis,  56 

and  spreading  of  disease,  59 
experimental    demonstration    of, 

57 

in  actinia,  56 
three  stages  of,  59 
Phenolphthalein  indicator,  524 
Phase,  negative,  63 

positive,  63 
Phycomycetes,  399 
Pinkeye,  228 

Piroplasma  bigeminum,  581 
morphology  of,  583 
natural  mode  of  transmission 

of,  586 
number    of    blood    corpuscles 

infected,  584 
bovis,  discovery  of,  581 
canis,  593 
ovis,  593 
Piroplasmosis  in  cattle  in  East  Africa. 

585 

of  horses,  595 

Pirosoma.    See  Piroplasma. 
Placental      circulation     as    portal    of 
entrance  for  pathogenic  bacteria,  50 
Plague  infection,  spread  of,  among  man 

and  animals,  233 » 
in  man,  234 
in  rats,  231 
latent,  235 

vaccine  and  serumtherapy  for,  238 
Plasmodium    immaculatum    or    falci- 

parum,  575 
malarise,  574 
vivax,  573 
Plasmolysis,  40 
Plasmoptysis,  40 
Plastids,  531 
Plates,     preparation     of,     by     Koch's 

method,  144 
prepared   for  bacterial   counts   of 

milk,  498 

various  forms  of  colonies  on,  167 
Platinum  loop  and  needle,  101 

spoon  for  soil  examination,  182 
Pleuropneumonia,  contagious,  in  cattle, 

440 

bacteriology  of,  441 
pathological  lesions  of,  440 


Pleuropneumonia,  contagious, in  cattle, 
protective  inoculation  against, 
443 

in  horses,  streptococci  in,  205 
septic,  of  calves,  226 
Pneumobacillus  of  Friedlander,  379 
Pneumococcus  of  Friedlander,  379 
Pneumonomycosis  in  man  and  animals. 

417 

Polar  bodies,  staining  of,  113 
Pool  in  opsonic  work,  64 
Porospora  gigantea,  530 
Portals     of    entrance     of     pathogenic 

bacteria,  48 
Postmortem  examination,  bacteriologic 

143 
Potash,  permanganate,  as  a  disinfectant, 

188 
Potato  bacillus.     See  Bacillus  mesen- 

tericus  vulgatus. 
culture  media,  134 
Precipitin    test  of   blood,  medicolegal, 

70 

Precipitins,  69 
Proteosoma  danilewsky,  576 
Protozoa,  classification  of,  537 

definition  and  morphology  of,  529 
encysted  stage  of,  530 
metabolism  of,  536 
nucleus  and  nuclear  substances  of, 

531 

organs  of  locomotion  of,  531 
pathogenic,  529 
period  of  maturity  and  old  age  of, 

536 

reproduction  of,  532 
shape  and  size  of,  530 
structure  of,  530 
Pseudobranches  of  bacteria,  28 
Pseudodiphtheria  bacillus,  211 
Pseudofarcy.     See  Lymphangitis,  epi- 
zootic. 

Pseudofilaments,  formation  of,  34 
Pseudoglanders,  315 
Pseudopodia,  531 

of  ameba,  539 

'  Pseudo-Rauschbrand,"  266 
Pseudotuberculosis,  366 

of  sheep,  368 
Psittacosis  of  parrots,  294 
Psorospermium  cuniculi,  570 
Pulex,  role  of,  in  spreading  plague,  233 
Pure  cultures    by   preliminary   animal 

inoculations,  150 

Pustule,  malignant.    See  Anthrax. 
Putrefaction,  definition  of,  458 
pyelonephritis  bacillosa  bovum,  207 
Pyocyanin,  201 


QUARTER-ILL  of  cattle,  254 


GENERAL  INDEX 


641 


R 


RABBIT'S  serum,  sensitized  to  sheep's 

corpuscles,  77,  79 
Rabies,  consumptive  form  of,  602 

diagnosis    of,   based    upon    Negri 

bodies,  604 
differences    in    virulency   between 

street  and  fixed  virus,  616 
dumb,  601 
furious  type  of,  601 
historical  and  occurrence  of,  597 
how   to    prepare    dog's   brain   for 

laboratory  examination,  609 
immunization  against,  612 
natural  infection  with,  597 
new  observations  on  pathology  and 

pathogenesis  of,  599 
pathological  lesions  of,  603 
period  of  incubation  of,  598 
preparation  of  attenuated  virus  for, 

614 

recovery  of  dog  from,  602 
resistance  of  virus  of,  617 
spread  of  virus  in  infection  with, 

611 
symptoms  of,  in  dog,  600 

in  man,  602 
Van  Gehuchten  and  Nellis'  changes 

in  nerve  ganglia  in,  604 
Radium   emanations,    effect    of,    upon 

bacteria,  187 
Rainey's  tubules,  578 
Rat  leprosy,  pathology  and  bacteriology 

of,  373 
pathological   lesions   produced   by 

plague  bacillus  in,  231 
Ray^fungus.    See  Actinomycosis. 
Receptors,  free,  floating  in  serum,  75 

of  cells,  75 

Reflector  of  microscope,  88 
Reichel  bacteriologic  filter,  160 
Reindeer  plague,  266 
Rennet,  516  • 

ferment,  test  for,  139 
Respiratory  tract  as  a  portal  of  en- 
trance for  pathogenic  bacteria,  49 
Rhipicephalus  annulatus,  586 

decoloratus,  as  transmitter  of  spiro- 

chete  infection,  390 
Rinderpest.     See  Cattle  plague. 
Root  nodules  of  leguminosae,  461 
Rosolic-acid  indicator,  524 


SACCHAROMYCES  fragilis,  479 

lactis  acidi,  479 
Saccharomycpsis      farciminosus.      See 

Lymphangitis,  epizootic. 
Saprophyte,  pathogenic    or    toxigenic, 

455 

41 


Saprophytes  and  parasites,  definition  of, 

24 

strict  or  obligate,  24 
Sarcina  aurantiaca,  438 
definition  of,  33 
lutea,  437 
mobilis,  438 
rosacea,  479 
rubra,  438 
ventriculi,  438 
Sarcocystis  bertrami,  579 
lindemani,  579 
miescheriana,  579 
tenella,  579 
Sarcodina,  537 
Sarcophysema  hsemorrhagicum  bovis, 

254 

Sarcosporidia,  578 
Scarlet  fever,  spread  by  milk,  486 
Schizomycetes,  26 
Schlegel  method  of  staining  actinomy- 

cotic  tissue,  409 
Scours,  white,  in  calves,  292 
Sectioning  of  embedded  tissues,  117 
Sedgwick  and  Tucker  air-filtering  de- 
vice, 180 
Sensibility,  bacteriochemical.     See 

Chemotaxis. 

Septicemia,  hemorrhagic,  of  bo  vines,  224 
of  sheep,  226 
of  swine,  226 

in  chickens,  streptococci  in,  205 
pluriformis  or  polymorpha,  221 
Septicemias  of  birds,  223 

various,  of  bovines,  226 
Serum,  antirabic,  617 
bouillon,  133 
hemolytic,  inactivated,  77 

reactivation  of,  77 
Shake  cultures,  150 

various  characteristics  of,  166 
Sheep's  red  blood  corpuscles,  washed.  79 
Side-chains  and  immunity,  74 
haptophile,  75 
haptophore,  74 

in  chemical  considerations,  74 
toxophile,  75 
toxpphore,  75 
Signet-ring  form  of  malarial  organisms, 

575 

Silkworm  disease,  577 
Skin  as  a  portal  of  entrance  for  patho- 
genic bacteria,  48 
Sleeping  sickness,  African,  in  man,  556, 

559,  562 

Slide  and  covers,  100 
Smegma  bacillus,  372 
Soil  bacteria,  great  resistance  of  spores 

of,  186 

bacteriologic  examination  of,  181 
Spindle,  achromatic,  533 
Spiradenitis  coccidiosa,  571 
Spirilla,  383 

in  water,  386 


642 


GENERAL  INDEX 


Spirillum,  general  characteristics  of,  26  , 
of  Asiatic  cholera,  385 

in  milk,  486 
Obermeieri,  387 
of  Denecke,  386 
of  Finkler  and  Prior,  386 
of  Metchnikoff,  383 
Theileri,  390 
Spirocheta  anserina,  389 
balanitidis,  389 
buccalis,  389 
dentium,  389 
Duttoni,  387 
gracilis,  389 
pallida,  387 

method  of  staining  of,  388 
pseudopallida,  389 
refringens,  389 
Spirochete,  387 

classification  of,  as  bacteria  and  not 

as  protozoa,  390 
demonstration    of,  by  blackening 

background,  115 
general  characteristics  of,  26 
in  birds,  389 
in  mammals,  390 
in  man,  387 
Spironema  pallida,  387 
Spongioplasm,  protozoan,  530 
Spontaneous  generation,  17,  18 
Sporangium,  399 
Spore  formation,  35 

in  hyphomycetes,  399 
in  protozoa,  535 
Spores,  germination  of,  36 

morphology  and  biologic   proper- 
ties of,  35 
of    hyphomycetes,    resistance    of, 

400 
staining  of,  110 

Klein's  method,  111 
Moeller's  method,  110 
Sporoblast,  535 
Sporozoa,  538 

definition  of  subphylum,  569 
Sporozoite,  535 
Sporulation,  35 
Stab  cultures,  how  made,  149 

various  characteristics  of,  164, 165 
Staining  of  sections,  119 
Stains,  anilin,  preparation  of  solutions 

of,  105 

Standard  solutions,  empirical,  524 
Staphylococci.  definition  of,  33 

of  Lucet  as  cause  of  mastitis  in 

cows,  489 

Staphylococcus  mastitidis,  489 
pyogenes,  194 
bovis,  207 

cultural  and  biological  prop- 
erties of,  196 
experiments  with,  197 
morphology  and  staining  prop- 
erties of,  195 


Staphylococcus  pyogenes,   occurrence 

and  pathogenesis  of,  195 
resistance  of,  197 
vaccine  therapy,  197 
varieties  of,  195 

Starter  in  preparation  of  butter,  513,514 
Steam  sterilizer,  125 
Sterilization,  meaning  of  term,  44 
Sterilizer,  dry  heat,  126 

steam,  126 

Stewart's  method  of  estimating  leuko- 
cytes in  milk,  490 
Stick  cultures  for  anaerobic  bacteria, 

151 

Storch's  test  for  enzymes  in  milk,  508 
Streak  cultures,  how  made,  149 

various  characteristics  of,  166 
Streptococci,  definition  of,  33 

in     apoplectiform     septicemia     in 

chickens,  205 
in  contagious  pleuropneumonia  of 

horses,  205 

in  morbus  maculosus  equorum,  205 
Streptococcus  coli  brevis,  474 

mirabilis,  474 
equi,  204 
Guentheri,  474 
lacticus  of  Kruse,  474 

role  of,  in  souring  of  cream,  513 
lactis  inocuus,  474 

Kefir,  474 
mastiditis,  474,  488 
mirabilis  of  Roscoe,  474 
of  abortion  in  mares,  321 
of  vaginitis  verrucosa  of  cows,  322 
pyogenes,  198 
bovis,  207 

cultural  and  biologic  proper- 
ties of,  199 

morphology  and  staining  prop- 
erties of,  198 
occurrence  and  pathogenesis 

of,  198 

resistance  of,  200 
vaccine  therapy,  200 
Streptothrices,  pathogenic,  402 
Streptothrix,  400 

acid-fast,  in  man,  404 
canis,  403 
caprae,  403 
cuniculi,  212 
farcinica,  402 
necrophora,  212 
Strauss  test  for  glanders,  310 
Subcultures,  how  made,  149 
Subcutaneous  inoculation,  170 
Sublimate,  corrosive,  as  disinfectant,  188 
Suction  pump  for  bacterial  filters,  161, 

162 

Sugar  agar,  132 
gelatin,  132 

Sugars,  quantitative  estimation  of,  525 
Sulphur  as  disinfectant,  191 
Sunlight,  effect  upon  bacteria,  44,  187 


GENERAL  INDEX 


643 


Suppuration,  193 

Surra,  558 

Susceptibility,  individual,  54 

Swine  erysipelas,  natural  infection  with, 

300 
protective  inoculation  against, 

301 

plague,  226 
Symbiosis  and  symbiotes,  25,  43 


TEMPERATURE  limits  for  bacterial  life, 

38 

maximum  and  minimum  of  bacte- 
rial growth,  38 
optimum  of  bacteria,  38 
tests  for  effect  upon  bacteria,  185 
Test-tubes,  filling  with  culture  media, 

127 

Tetanus  in  horse  and  man,  271 
in  laboratory  animals,  272 
preparation  of  antitoxin  for,  274 
toxin,  272 

Tetrads,  definition  of,  33 
Texas  fever  of  cattle/581 

diagnosis  of,  583 
epidemiology  of,  589 
immunization  against,  590 
pathological  anatomy  of, 

582 

Thallus  of  hyphomycetes,  398 
Thermometer  scale  of  Celsius,  Fahren- 
heit, and  Reaumur,  623 
Thermoregulator,  157 

how   constructed   and   how   regu- 
lated, 158 
Thermostat,  156 

Thoma-Zeiss  counting  chamber  in  esti- 
mation of  leukocytes  in  milk,  491 
Tissue  bacterioid  of  Bacillus  radicicola, 

463 
Tissues,  fixing  of,  115 

methods  of  embedding  of,  116 
Torula,  a  genus  of  blastomyces,  426 
cause  of  tumor  in  horse,  430 
lactis,  479 
Toxins  and  antitoxins,  69 

extracellular  and  intracellular,  53 
relation  of,  to  disease,  53 
soluble  and  insoluble  53 
Toxoids,  definition  and  mode  of  forma- 
tion of,  75 

Transplanting  of  pure  cultures,  149 
Trembles  of  cattle,  393 
Treponema  pallidum,  387 
Trichomonas,  564 
Trichophyton  ectothrix,  419 
endo-ectothrix,  419 
tonsurans,  418 
Trommsdorf's    method    of    estimating 

leukocytes  in  milk,  490 
Trophonucleus,  531 


Tropical  ulcer,  Wright's  intracorpuscu- 

lar  bodies  in,  567 

Trypanosoma  Americanum,  n.  sp.,  562 
Brucei,  558 
classification  and  morphology  of, 

553 

equinum,  558 
equiperdum,  558 
Evansii,  558 
habitat  of,  555 

how  to  obtain  them  in  pure  cul- 
tures, 556 

infection,  how  propagated,  560 
in  birds,  563 
latent,  561 
pathological  changes  due  to, 

557 

method  of  examining  for,  555 
stage  in  life  cycle  of  halteridium,  563 

of  piroplasma,  563 
Theileri,  558 
Trypanosomes  and  trypanosomiases,  553 

pathogenic,  558 
Tsetse  fly,  560 
Tubercle,  structure  of,  331 
Tubercles,  solitary,  335 
Tuberculin,  injection  of,  reaction  after, 

354 

Koch's  old,  352 
test,  cutaneous,  357 
tests,  352 
Tuberculosis,  324 

among  domestic  animals,  prophy- 
laxis and  eradication  of,  359 
avian,  343,  364 

transmission  of,  to  mammals, 

364 

bovine,  transmissibility  of,  to  man, 
German  collective  investigation, 
483 
discovery  of  etiology  of,  by  Koch, 

326 

distribution  of,  in  man,  326 
first  inoculation  experiments  with, 

325 

histopathology  of,  331 
historical,  324 
hyperplastic,  335 
in  animals,  327 

in  man,  due  to  bovine  type  of  ba- 
cilli, 363 
miliary,  336 

general,  acute,  337 
modes  of  infection  and  transmis- 
sion in,  328 

of  fish  and  turtles,  344 
of  food-producing  animals,  343 
of  parrots,  344 

organs  affected  by,  in  cattle,  338 
in  hogs,  340 
in  man,  337 

prevention  of,  among  swine,  360 
process  of  caseation  in,  333 
protective  inoculation  against,  357 


644 


GENERAL  INDEX 


Tuberculosis,  transmission  of,   bypla- 

cental  circulation,  330 
by  sexual  intercourse,  330 
TyndalPs  method  of  fractional  steriliza- 
tion, 125 
Typhoid  fever,  equine,  228 

-like  bacillus  of  Lustig,  473 
Tyrothrix  distortus  479 
geniculatus,  479 
in  ripening  of  cheese,  517 
tenuis,  479 


ULTRAMICROSCOPIC  viruses,  440 
Unna's  alkaline  methylene  blue,  120 
Urea,  fermentation  of,  458 
Urobacillus  Pasteuri,  459 
Ushinsky's  solution,  136 


VACCINES  and  antitoxic  sera,  prepara- 
tion of,  71 

autogenic,  preparation  of,  66 

for  raising  opsonic  index,  62 
Van  Leewenhoeck's  discoveries,  18 
Varo's  theory  of  disease,  17 
Vibrio  berolinensis,  386 

danubius,  386 

general  characteristics  of,  26 

Metchnikovi,  383 

of  Asiatic  cholera,  385 

proteus,  386 

Schuylkiliensis,  386 

tyrogenum,  386 
Vibriones,  pathogenic,  383 


Vinegar,  manufacture  of,  467 

mother  of,  466 
Virulence  or  virulency,  54 

increase  of,  54 

lessening  of,  55 

Virus,  invisible,  of  hog  cholera,  281 
Viruses,  ultramicroscopic,  as  cause  of 
disease,  440 

W 

WASSERMANN  serum  test  for  syphilis,  78 

Water,  bacteriologic  examination  of, 
180 

Weigert's  method,  applied  to  actinomy- 
cotic  tissue,  409 

Widal-Gruber  agglutination  test  for  ty- 
phoid fever,  287 

Winogradsky's  culture  media,  136 

Wolffhuegel  counting  apparatus,  174 

Wooden  tongue  of  cattle  in  actinomy- 
cosis,  406 

Woolsorter's  disease.     See  Anthrax. 

Wort  gelatin  and  agar,  134 

Wound  infection,  193 

Wright's  eosin-methylene-blue  stain,  113 
methods  for  anaerobic  cultures,  153 


X-RAYS,  effect  of,  upon  bacteria,  187 


Z 


ZIEHL'S  carbol  fuchsin,  109 
Zooglea,  30 
Zygospores,  400 


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